Antibodies that block receptor protein tyrosine kinase activation, methods of screening for and uses thereof

ABSTRACT

Molecules comprising the antigen-binding portion of antibodies that block constitutive and/or ligand-dependent activation of a receptor protein tyrosine kinase, such as fibroblast growth factor receptor 3 (FGFR3), are found through screening methods, where a soluble dimeric form of a receptor protein tyrosine kinase is used as target for screening a library of antibody fragments displayed on the surface of bacteriophage. The molecules of the present invention which block constitutive activation can be administered to treat or inhibit skeletal dysplasia, craniosynostosis disorders, cell proliferative diseases or disorders, or tumor progression associated with the constitutive activation of a receptor protein tyrosine kinase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/IL02/00494 filed Jun. 20, 2002, the content of which is expresslyincorporated herein by reference thereto, and which claims the benefitof U.S. Provisional Application No. 60/299,187 filed Jun. 20, 2001.

FIELD OF THE INVENTION

The present invention relates to immunoglobulins and functionalfragments thereof useful for blocking activation of receptor proteintyrosine kinases, methods for screening for such immunoglobulins,compositions comprising said immunoglobulins and methods of using thesame for treating or inhibiting disease, such as skeletal dysplasia,craniosynostosis disorders, cell proliferative diseases or disorders, ortumor progression.

BACKGROUND OF THE INVENTION

A wide variety of biological processes involves complex cellularcommunication mechanisms. One of the primary means of continual exchangeof information between cells and their internal and externalenvironments is via the secretion and specific binding of peptide growthfactors. Growth factors exert pleiotropic effects and play importantroles in oncogenesis and the development of multicellular organismsregulating cell growth, differentiation and migration. Many of thesefactors mediate their effects by binding to specific cell surfacereceptors. The ligand-activated receptors start an enzymatic signaltransduction cascade from the cell membrane to the cell nucleus,resulting in specific gene regulation and diverse cellular responses.

Protein Kinases

One of the key biochemical mechanisms of signal transduction involvesthe reversible phosphorylation of proteins, which enables regulation ofthe activity of mature proteins by altering their structure andfunction.

Protein kinases (“PKs”) are enzymes that catalyze the phosphorylation ofhydroxy groups on tyrosine, serine and threonine residues of proteins.The consequences of this seemingly simple activity are staggering; cellgrowth, differentiation and proliferation; e.g., virtually all aspectsof cell life in one way or another depend on PK activity. Furthermore,abnormal PK activity has been related to a host of disorders, rangingfrom relatively non-life threatening diseases such as psoriasis toextremely virulent diseases such as glioblastoma.

The kinases fall largely into two groups, those specific forphosphorylating serine and threonine, and those specific forphosphorylating tyrosine. Some kinases, referred to as “dualspecificity” kinases, are able to phosphorylate tyrosine as well asserine/threonine residues.

Protein kinases can also be characterized by their location within thecell. Some kinases are transmembrane receptor proteins capable ofbinding ligands external to the cell membrane. Binding the ligandsalters the receptor protein kinase's catalytic activity. Others arenon-receptor proteins lacking a transmembrane domain and yet others areecto-kinases that have a catalytic domain on the extracellular (ecto)portion of a transmembrane protein or which are secreted as solubleextracellular proteins.

Many kinases are involved in regulatory cascades where their substratesmay include other kinases whose activities are regulated by theirphosphorylation state. Thus, activity of a downstream effector ismodulated by phosphorylation resulting from activation of the pathway.

Receptor protein tyrosine kinases (RPTKs) are a subclass oftransmembrane-spanning receptors endowed with intrinsic,ligand-stimulatable tyrosine kinase activity. RPTK activity is tightlycontrolled. When mutated or altered structurally, RPTKs can becomepotent oncoproteins, causing cellular transformation. In principle, forall RPTKs involved in cancer, oncogenic deregulation results from reliefor perturbation of one or several of the auto-control mechanisms thatensure the normal repression of catalytic domains. More than half of theknown RPTKs have been repeatedly found in either mutated oroverexpressed forms associated with human malignancies (includingsporadic cases; Blume-Jensen et al., 2001). RPTK overexpression leads toconstitutive kinase activation by increasing the concentration ofdimers. Examples are Neu/ErbB2 and epidermal growth factor receptor(EGFR), which are often amplified in breast and lung carcinomas and thefibroblast growth factors (FGFR) associated with skeletal andproliferative disorders (Blume-Jensen et al., 2001).

Fibroblast Growth Factors

Normal growth, as well as tissue repair and remodeling, require specificand delicate control of activating growth factors and their receptors.Fibroblast Growth Factors (FGFs) constitute a family of over twentystructurally related polypeptides that are developmentally regulated andexpressed in a wide variety of tissues. FGFs stimulate proliferation,cell migration and differentiation and play a major role in skeletal andlimb development, wound healing, tissue repair, hematopoiesis,angiogenesis, and tumorigenesis (reviewed in Ornitz and Itoh, 2001).

The biological action of FGFs is mediated by specific cell surfacereceptors belonging to the RPTK family of protein kinases. Theseproteins consist of an extracellular ligand binding domain, a singletransmembrane domain and an intracellular tyrosine kinase domain whichundergoes phosphorylation upon binding of FGF. The FGF receptor (FGFR)extracellular region contains three immunoglobulin-like (Ig-like) loopsor domains (D1, D2 and D3), an acidic box, and a heparin binding domain.Five FGFR genes that encode for multiple receptor protein variants havebeen identified to date.

Another major class of cell surface binding sites includes binding sitesfor heparan sulfate proteoglycans (HSPG) that are required for highaffinity interaction and activation of all members of the FGF family.Tissue-specific expression of heparan sulfate structural variants conferligand-receptor specificity and activity of FGFs.

FGFR-Related Disease

Recent discoveries show that a growing number of skeletal abnormalities,including achondroplasia, the most common form of human dwarfism, resultfrom mutations in FGFRs. Specific point mutations in different domainsof FGFR3 are associated with autosomal dominant human skeletal disordersincluding hypochondroplasia, severe achondroplasia with developmentaldelay and acanthosis nigricans (SADDAN) and thanatophoric dysplasia (TD)(Cappellen et al., 1999; Webster et al., 1997; Tavormina et al.,1999).FGFR3 mutations have also been described in two craniosynostosisphenotypes: Muenke coronal craniosynostosis (Bellus et al., 1996; Muenkeet al., 1997) and Crouzon syndrome with acanthosis nigricans (Meyers etal., 1995). Crouzon syndrome is associated with specific point mutationsin FGFR2 and both familial and sporadic forms of Pfeiffer syndrome areassociated with mutations in FGFR1 and FGFR2 (Galvin et al., 1996;Schell et al., 1995). Mutations in FGFRs result in constitutiveactivation of the mutated receptors and increased receptor proteintyrosine kinase activity, rendering cells and tissue unable todifferentiate. Specifically, the achondroplasia mutation results inenhanced stability of the mutated receptor, dissociating receptoractivation from down-regulation, leading to restrained chondrocytematuration and bone growth inhibition (reviewed in Vajo et al., 2000).

There is accumulating evidence for mutations activating FGFR3 in varioustypes of cancer. Constitutively activated FGFR3 in a large proportion oftwo common epithelial cancers, bladder and cervix, as well as inmultiple myeloma, is the first evidence of an oncogenic role for FGFR3in carcinomas. FGFR3 currently appears to be the most frequently mutatedoncogene in bladder cancer where it is mutated in almost 50% of thecases and in about 70% of cases having recurrent superficial bladdertumors (Cappellen, et al, 1999; van Rhijn, et al, 2001; Billerey, et al,2001). FGFR3 mutations are seen in 15-20% of multiple myeloma caseswhere point mutations that cause constitutive activation directlycontribute to tumor development and progression (Chesi, et al, 1997;Plowright, et al, 2000, Ronchetti, et al, 2001).

In this context, the consequences of FGFR3 signaling appear to be celltype-specific. In chondrocytes, FGFR3 hyperactivation results in growthinhibition (reviewed in Ornitz, 2001), whereas in the myeloma cell itcontributes to tumor progression (Chesi et al., 2001).

In view of the link between RPTK-related cellular activities and anumber of human disorders various strategies have been employed totarget the receptors and/or their variants for therapy. Some of thesehave involved biomimetic approaches using large molecules patterned onthose involved in the cellular processes, e.g., mutant ligands (U.S.Pat. No. 4,966,849); soluble receptors and antibodies (WO 94/10202, U.S.Pat. No. 6,342,219); RNA ligands (U.S. Pat. No. 5,459,015) and tyrosinekinase inhibitors (WO 94/14808; U.S. Pat. No. 5,330,992).

Antibody Therapy

The search for new therapies to treat cancer and other diseasesassociated with growth factors and their corresponding cell surfacereceptors has resulted in the development of humanized antibodiescapable of inhibiting receptor function. For example, U.S. Pat. Nos.5,942,602 and 6,365,157 disclose monoclonal antibodies specific for theHER2/neu and VEGF receptors, respectively. U.S. Pat. No. 5,840,301discloses a chimeric, humanized monoclonal antibody that binds to theextracellular domain of VEGF and neutralizes ligand-dependentactivation. U.S. Pat. No. 5,707,632 discloses a method for producing anantibody to a FGFR and a monoclonal antibody to FGFR that blocks bindingof fibroblast growth factor to said fibroblast growth factor receptorsequences.

There remains an unmet need for highly selective molecules capable ofblocking aberrant constitutive receptor protein tyrosine kinaseactivity, in particular FGFR activity, thereby addressing the clinicalmanifestations associated with the above-mentioned mutations, andmodulating various biological functions.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

In one aspect the present invention provides molecules which are able toblock receptor protein tyrosine kinase (RPTK) activity.

In another aspect the present invention provides molecules which areable to block fibroblast growth factor receptor (FGFR) activity,preferably fibroblast growth factor receptor 3 (FGFR3) activity.

In yet another aspect the present invention provides polypeptidesencoding molecules which are able to block receptor protein tyrosinekinase (RPTK) activity, preferably FGFR activity, and more preferablyFGFR3 activity.

In a further aspect the present invention provides a method to screenfor molecules which are able to block receptor protein tyrosine kinaseactivity.

In one aspect the present invention provides a pharmaceuticalcomposition comprising as an active ingredient a therapeuticallyeffective amount of molecules of the invention useful in treating orpreventing skeletal and proliferative diseases and disorders.

In another aspect the present invention provides a method for inhibitinggrowth of tumor cells associated with ligand-dependent or constitutiveactivation of a RPTK. According to one embodiment the RPTK is afibroblast growth factor receptor. According to another embodiment theRPTK is FGFR3.

In yet a further aspect the present invention provides a method fortreating skeletal disorders associated with ligand-dependent orconstitutive activation of a RPTK. According to one embodiment the RPTKis a fibroblast growth factor receptor. According to another embodimentthe RPTK is FGFR3.

In one aspect the present invention provides a method for blockingreceptor protein tyrosine kinase activation in the cells of patients inneed thereof by treatment with molecules capable of inhibiting receptorprotein tyrosine kinase function.

In yet another aspect the present invention provides a method forinhibiting tumor growth, tumor progression or metastases.

In yet another aspect the present invention provides molecules usefulfor in vivo imaging of diseased states. In yet another aspect thepresent invention provides a kit comprising molecules of the invention.For example, a kit would comprise an antigen binding molecule of theinvention and at least one reagent suitable for detecting the presenceof said molecule when bound to said receptor protein tyrosine kinase andinstructions for use.

These and other aspects are met by the invention disclosed herein.

The present invention provides a molecule comprising the antigen-bindingportion of an antibody which has a specific affinity for a receptorprotein tyrosine kinase (RPTK) and which blocks ligand-independent(constitutive activation) of a receptor protein tyrosine kinase. Thepresent invention further provides a molecule that comprises theantigen-binding portion of an antibody which has a specific affinity fora receptor protein tyrosine kinase and which blocks ligand-dependentactivation of a fibroblast growth factor receptor (FGFR), includingFGFR1 and FGFR3.

Certain mutations in the genes of receptor protein tyrosine kinasesresult in activation of the receptor in a manner that is independent ofthe presence of a ligand. Such ligand-independent, or constitutive,receptor protein tyrosine kinase activation results in increasedreceptor activity. The clinical manifestations of certain mutations infibroblast growth factor receptors (FGFR) are skeletal and proliferativedisorders and diseases, including achondroplasia and various cancers.

Specific molecules of the present invention were found to inhibit orblock constitutive activation of the FGFR3. Generation of inhibitorymolecules would be useful for developing medicaments for use in treatingor preventing skeletal and proliferative diseases and disordersassociated with constitutive activation of receptor protein tyrosinekinases.

The present invention is directed to novel molecules comprising anantigen binding domain which binds to a receptor protein tyrosine kinaseand blocks constitutive activation of said receptor protein tyrosinekinase. The molecules of the invention maybe antibodies or antigenbinding fragments thereof.

One embodiment of the present invention provides a molecule which bindsto the extracellular domain of a receptor protein tyrosine kinase andblocks constitutive and ligand-dependent activation of the receptor.

A currently more preferred embodiment of the present invention providesa molecule which binds to the extracellular domain of an FGF receptorand blocks constitutive and ligand-dependent activation of the receptor.

A currently most preferred embodiment of the present invention providesa molecule which binds FGFR3 and blocks constitutive andligand-dependent activation of the receptor, comprising V_(L)-CDR3 andV_(H)-CDR3 regions having amino acid SEQ ID NO:25 and SEQ ID NO:24,respectively and the corresponding isolated nucleic acid moleculescomprising polynucleotide sequence SEQ ID NO:51 and SEQ ID NO:50.

A currently most preferred embodiment of the present invention providesa molecule which binds FGFR3 and blocks constitutive andligand-dependent activation of the receptor, comprising V_(L)-CDR3 andV_(H)-CDR3 regions having SEQ ID NO:13 and SEQ ID NO:12 or SEQ ID NO:9and SEQ ID NO:8, respectively and the corresponding isolated nucleicacid molecules comprising polynucleotide sequence SEQ ID NO:35 and SEQID NO:34 or SEQ ID NO:31 and SEQ ID NO:30.

Another currently preferred embodiment of the present invention providesa molecule herein denoted MSPRO12 comprising a variable light chain(V_(L)) having SEQ ID NO:87 and a variable heavy chain (V_(H)) havingamino acid SEQ ID NO:98 and the corresponding isolated nucleic acidmolecules comprising polynucleotide sequences having SEQ ID NO:68 andSEQ ID NO:82, respectively.

Another currently preferred embodiment of the present invention providesa molecule herein denoted MSPRO2 comprising a variable light chain(V_(L)) having SEQ ID NO:85 and a variable heavy chain (V_(H)) havingSEQ ID NO:96 and the corresponding isolated nucleic acid moleculescomprising polynucleotide sequences having SEQ ID NO:67 and SEQ IDNO:77.

A currently most preferred embodiment of the present invention providesa molecule, herein denoted MSPRO59, comprising a variable light chain(V_(L)) having SEQ ID NO:95 and a variable heavy chain (V_(H)) havingSEQ ID NO:106 having the corresponding isolated nucleic acid moleculescomprising polynucleotide sequences having SEQ ID NO:69 and SEQ IDNO:84, respectively.

According to the principles of the present invention, molecules whichbind FGFR and block ligand-dependent receptor activation are provided.These molecules are useful in treating disorders and diseases associatedwith an FGFR that is activated in a ligand-dependent manner includingcertain skeletal disorders, hyperproliferative diseases or disorders andnon-neoplastic angiogenic pathologic conditions such as neovascularglaucoma, macular degeneration, hemangiomas, angiofibromas, psoriasisand proliferative retinopathy including proliferative diabeticretinopathy.

In one embodiment the present invention provides a molecule which bindsFGFR3 and blocks ligand-dependent activation of the receptor, comprisingV_(H)-CDR3 and V_(L)-CDR3 regions having SEQ ID NO:20 and SEQ ID NO:21,respectively and the corresponding polynucleotide sequence having SEQ IDNO:44 and SEQ ID NO:45, respectively. In another embodiment the presentinvention provides a molecule comprising a variable light chain (V_(L))having SEQ ID NO:92 and a variable heavy chain (V_(H)) having SEQ IDNO:103, having the corresponding isolated nucleic acid moleculescomprising polynucleotide sequences having SEQ ID NO:58 and SEQ IDNO:80, respectively.

Other embodiments of the present invention provide a molecule whichbinds FGFR3 and blocks ligand-dependent activation of the receptor,comprising V_(H)-CDR3 and V_(L)-CDR3 regions selected from SEQ ID NO:10and SEQ ID NO:11; SEQ ID NO:14 and SEQ ID NO:15; SEQ ID NO:16 and SEQ IDNO:17; SEQ ID NO:18 and SEQ ID NO:19; SEQ ID NO:26 and SEQ ID NO:27 andSEQ ID NO:28 and SEQ ID NO:29 and the corresponding isolated nucleicacid molecules comprising polynucleotide sequences having SEQ ID NOaccording to Table 1B.

Additional embodiments of the present invention provide molecules havingan antigen binding domain comprising a V_(L) region and a V_(H) region,respectively, selected from SEQ ID NO:86 and SEQ ID NO:97; SEQ ID NO:88and SEQ ID NO:99; SEQ ID NO:89 and SEQ ID NO:100; SEQ ID NO:90 and SEQID NO:101; SEQ ID NO:91 and SEQ ID NO:102; SEQ ID NO:92 and SEQ IDNO:103; and SEQ ID NO:94 and SEQ ID NO:105 and the correspondingisolated nucleic acid molecules comprising polynucleotide sequenceshaving SEQ ID NO:63 and SEQ ID NO:78; SEQ ID NO:60 and SEQ ID NO:71; SEQID NO:57 and SEQ ID NO:72; SEQ ID NO:64 and SEQ ID NO: 79 SEQ ID NO:55and SEQ ID NO:73; SEQ ID NO:58 and SEQ ID NO:80; and SEQ ID NO:62 andSEQ ID NO:76.

One embodiment of the present invention provides a molecule comprisingV_(H)-CDR3 and V_(L)-CDR3 domains of amino acid sequences having SEQ IDNO:22 and SEQ ID NO:23, which has specific affinity for FGFR1 and whichblocks ligand-dependent activation of FGFR1, and the correspondingpolynucleotide sequences having SEQ ID NO:46 and SEQ ID NO:47.

Another embodiment of the present invention provides a moleculecomprising V_(H) and V_(L) domains of amino acid sequences having SEQ IDNO:104 and 93, which has specific affinity for FGFR1 and which blocksligand-dependent activation of FGFR1, and the corresponding isolatednucleic acid molecules comprising polynucleotide sequences having SEQ IDNO:75 and SEQ ID NO:66.

The present invention also relates to methods for screening for themolecules according to the present invention by using a dimeric form ofa receptor protein tyrosine kinase as a target for screening a libraryof antibody fragments.

According to one currently preferred embodiment the screening methodcomprises

-   -   providing a library of of antigen binding fragments;    -   screening said library for binding to a dimeric form of a        receptor protein tyrosine kinase;    -   identifying an antigen binding fragment which binds to the        dimeric form of the receptor protein tyrosine kinase as a        candidate molecule for blocking constitutive activation of the        receptor protein tyrosine kinase; and    -   determining whether the candidate molecule blocks constitutive        and or ligand-dependent activation of the receptor protein        tyrosine kinase in a cell.

According to another embodiment, the dimeric form of the RPTK is asoluble extracellular domain of a receptor protein tyrosine kinase.Non-limiting examples of receptor protein tyrosine kinases disclosed inBlume-Jensen et al. (2001) include EGFR/ErbB1, ErbB2/HER2/Neu,ErbB/HER3, ErbB4/HER4, IGF-1R, PDGFR-a, PDGFR-β, CSF-1R, kit/SCFR,Flk2/FH3, Flk1/VEGFR1, Flk1/VEGFR2, Flt4/VEGFR3, FGFR1, FGFR2/K-SAM,FGFR3, FGFR4, TrkA, TrkC, HGFR, RON, EphA2, EphB2, EphB4, Axl, TIE/TIE1,Tek/TIE2, Ret, ROS, Alk, Ryk, DDR, LTK and MUSK. Heterodimeric form ofthe receptors my be used as antigen.

By using a dimeric form of the RPTK as bait in the screen, a moleculewhich would bind to the dimeric form of the receptor has beenidentified. This presents a novel concept in screening for antibodies orfragments thereof with the capacity to bind to a constitutivelyactivated RPTK such as those associated with various disorders anddiseases. It also presents an opportunity to screen for molecules whichbind to a heterodimer RPTK. A further aspect of the present inventionprovides a pharmaceutical composition comprising as an active ingredienta therapeutically effective amount of a molecule of the presentinvention in a pharmaceutically acceptable carrier or excipient usefulfor preventing or treating skeletal or cartilage diseases or disordersand craniosynostosis disorders associated with constitutive orligand-dependent activation of a receptor protein tyrosine kinase.

In one embodiment the pharmaceutical compositions of the presentinvention may be used for treating or preventing skeletal disordersassociated with aberrant FGFR signaling, including achondroplasia,thanatophoric dysplasia, Apert or Pfeiffer syndrome and relatedcraniosynostosis disorders.

A further aspect of the present invention provides a pharmaceuticalcomposition comprising as an active ingredient a therapeuticallyeffective amount of a molecule of the present invention in apharmaceutically acceptable carrier or excipient useful for preventingor treating cell proliferative diseases or disorders or tumorprogression, associated with the constitutive or ligand-dependentactivation of a receptor protein tyrosine kinase.

In one embodiment the pharmaceutical compositions of the presentinvention may be used for treating or preventing proliferative diseasesassociated with aberrant FGFR signaling, including multiple myeloma,transitional cell carcinoma of the bladder, mammary and cervicalcarcinoma, chronic myeloid leukemia and osteo- or chondrosarcoma.

A further aspect of the invention provides molecules comprising anantigen binding domain which can be conjugated to a cytotoxin useful fortargeting cells expressing said antigen. Another aspect of the presentinvention provides molecules comprising an antigen binding domain whichcan be conjugated to appropriate detectable imaging moiety, useful forin vivo tumor imaging.

A still further aspect of the present invention provides methods fortreating or inhibiting the aforementioned diseases and disorders byadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising a molecule of the present invention to a subjectin need thereof.

Other aspects of the invention will be apparent upon consideration ofthe following description, figures and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows hFR3²³⁻³⁷⁴TDhis purification by Coommassie stainedSDS-PAGE.

FIG. 2 shows hFR3²³⁻³⁷⁴TDhis binding to heparin and FGF9.

FIG. 3 shows the purification of FR3exFc and FR1exFc on SDS-PAGE.

FIG. 4 shows the neutralization effect of the hFR3²³⁻³⁷⁴TDhis andFR3exFc soluble receptors in a ligand-dependent proliferation assay.

FIG. 5 shows the effect of MS-PRO Fabs on proliferation ofFGFR-expressing cells.

FIG. 6 shows the effect of MSPRO Fabs on proliferation ofFGFR3-expressing cells.

FIGS. 7A and 7B show the neutralizing activity of several MSPRO Fabs ina proliferation assay using the FDCP-FR3 (C10; FIG. 7A) or the FDCP-FR1cells (FIG. 7B).

FIG. 8 shows the receptor specificity of MSPRO Fabs on RCJ cellsexpressing R3, ach, R1 or R3 receptors by Western blot using ananti-P-JNK (phosphorylated/activated Jun kinase) antibody. FIG. 8A showsdifferent MSPRO Fabs while FIG. 8B shows a dose response of MSPRO 12, 29and 13 on RCJ-FR3 cells.

FIG. 9 demonstrates the specificity and potency of MS-PRO Fabs byWestern blot with anti-P-ERK (phosphorylated/activated ERK) antibody.FIG. 9A shows a dose response of MSPRO 29, 59 and 54 on RCJ-M14 cells.FIG. 9B shows a dose response of MSPRO 29, 59 and 54 on RCJ-W11 cells.FIG. 9C shows a dose response of MSPRO 29, 59 and 54 on RCJ-R1-1 cells.FIG. 9D shows a dose response of MSPRO 29, 59 and 54 on RCJ-R2-2 cells.

FIG. 10 shows a diagrammatic representation of FGFR3 and of FGFR3truncations (D2-3, D2) and isoforms (IIIb, IIIc). The isoform IIIbdiffers from IIIc at the carboxy terminus of the IgIII domain asindicated with a dotted line.

FIG. 11 shows that the FGFR3 neutralizing Fabs recognize IgII or IgIIand III in the extracellular region of FGFR3.

FIG. 12 shows the proliferation level of FGFR3IIIb and FGFR3IIIcexpressing cells in the presence of MSPRO29. MSPRO 29 specificallyrecognizes the IIIc isoform of FGFR3.

FIGS. 13A and 13B show the results of a proliferation assay forFDCP-FR3IIIb or FDCP-FR3IIIc cells incubated with increasing doses ofthe indicated Fabs.

FIG. 14 shows iodinated MSPRO29 binding to FGFR3.

FIG. 15 shows results of a proliferation assay is a graph whereiniodinated MSPRO29 retained its activity against FGFR3.

FIG. 16 shows the selective binding of radiolabeled MSPRO29 tohistological sections of growth plate. FIG. 16A shows Hematoxylin-eosinstaining of growth plate treated with radiolabeled MSPRO29 at ×100magnification. FIG. 16B shows radiomicroscopic sections of growth platetreated with radiolabeled MSPRO29 at ×100 magnification. FIG. 16C showsradiomicroscopic sections of growth plate treated with radiolabeledMSPRO29 at ×400 magnification. FIG. 16D shows Hematoxylin-eosin stainingof growth plate treated with radiolabeled Ly6.3 at ×100 magnification.FIG. 16E shows radiomicroscopic sections of growth plate treated withradiolabeled Ly6.3 at ×100 magnification. FIG. 16F showsradiomicroscopic sections of growth plate treated with radiolabeledLy6.3 at ×400 magnification.

FIGS. 17A and 17B show a proliferation assay of FDCP-FR3 (17A) andFDCP-FR3ach cells (17B) incubated with FGF9 and with increasing doses ofindicated Fabs.

FIG. 18B shows that MSPRO12 and MSPRO59 inhibit the ligand independentproliferation of FDCP-FR3ach cells. FIG. 18A shows analysis of theligand-dependent FDCP-FR3 wt cells.

FIG. 19 shows the restoration of cell growth by MS-PRO54 and MSPRO59.

FIG. 20 represents the growth rate of treated bone with MS-PRO 59.

FIG. 21 is a flow chart of the experimental protocol for assessingreceptor activation and signaling.

FIG. 22 shows ¹²⁵I-MSPRO59 localization to the FDCP-FR3ach derivedtumor.

FIG. 23 shows the effect of MSPRO59 on inhibiting ligand-independenttumor growth.

FIG. 24 shows the effect of MSPRO59 on inhibiting ligand-independenttumor growth.

FIG. 25A shows the effect of MSPRO59 on inhibiting ligand-independenttumor growth.

FIG. 25B shows scFv MSPRO59 blocking the proliferation of FDCP-FR3(S375C) cells.

FIG. 26 shows the effect of MSPRO59 single chain antibody on inhibitingligand-independent tumor growth.

FIG. 27 shows binding of Fab Miniantibodies to FGFR3-Fc and FGFR1-Fc(ELISA).

FIG. 28 is an example of a Fab expression vector, having SEQ ID NO:52,for use in accordance with the present invention.

FIG. 29 is an example of a phage display vector, having SEQ ID NO:53,for use in accordance with the present invention.

FIG. 30 depicts the polynucleotide sequences of the V_(L) and V_(H) ofMSPRO antibodies of the present invention SEQ ID NOS: 54-84.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that neutralizingantibodies that block ligand-dependent and ligand-independent activationof fibroblast growth factor receptor 3 (FGFR3), a receptor proteintyrosine kinase (RPTK), can be obtained by screening an antibody libraryagainst a dimeric form of the extracellular portion of FGFR3. Until thepresent invention, the present inventors are unaware of any success inobtaining neutralizing antibodies that block constitutive activation ofany RPTK including FGFR or ligand-dependent FGFR activation.

For convenience certain terms employed in the specification, examplesand claims are described herein.

The term “receptor protein tyrosine kinase” or “RPTK” as used herein andin the claims refers to a subclass of transmembrane-spanning receptorsendowed with intrinsic, ligand-stimulatable tyrosine kinase activity.RPTKs comprise a large family of spatially and temporally regulatedproteins that control many different aspects of growth and development.When mutated or altered structurally, RPTKs can undergo deregulation andbecome activated in a ligand-independent, or constitutive, manner. Incertain cases they become potent oncoproteins, causing cellulartransformation.

As used herein and in the claims the term “fibroblast growth factorreceptor” or “FGFR” denotes a receptor specific for FGF which isnecessary for transducing the signal exerted by FGF to the cellinterior, typically comprising an extracellular ligand-binding domain, asingle transmembrane helix, and a cytoplasmic domain having tyrosinekinase activity. The FGFR extracellular domain consists of threeimmunoglobulin-like (Ig-like) domains (D1, D2 and D3), a heparin bindingdomain and an acidic box. Alternative splicing of the FGF receptor mRNAsgenerates different variants of the receptors. Certain abbreviations areemployed herein including “FR3” for FGFR3 and “FR1” for FGFR1.

Molecules, including antibodies and fragments thereof, comprising anantigen binding domain to a receptor protein tyrosine kinase are highlynecessary for the treatment of various pathological conditions.

In the past, the laboratory of the present inventors encountereddifficulties in raising neutralizing antibodies against FGFR3. When micewere immunized with the soluble monomeric FGFR3 receptor, by the timethe antibody titers began to increase, the mice died. The experimentsperformed in the laboratory of the present inventors that failed toobtain anti-FGFR3 neutralizing antibodies in mice are presented in theExamples.

-   -   By using a soluble dimeric form of the extracellular domain of        the FGFR3 receptor to screen for antibodies, e.g., Fabs, that        bind from a phage display antibody library, the present        inventors were able to overcome a problem in the prior art for        which there was yet no solution and to obtain numerous high        affinity (K_(D)<10 nM) antibodies (Fabs) that bind FGFR3 and        interfere with ligand binding, thereby blocking ligand-dependent        activation of FGFR3 signaling. Very surprisingly, from among the        group of Fabs that block ligand-dependent activation, Fab        antibodies which also block ligand-independent (constitutive)        activation of FGFR3 by blocking signaling caused by constitutive        dimerization of FGFR3 were identified. To the best of the        present inventors' knowledge, these Fab antibodies, which block        constitutive activation of FGFR3, are the first antibodies        against any receptor protein tyrosine kinase that block        constitutive, ligand-independent activation/signaling.

Trastuzamab, an anti-human epidermal growth factor receptor 2 (HER2)antibody, was the first humanized monoclonal antibody approved fortherapeutic use. Its mode of action appears to be manifold, includingHER2 down regulation, prevention of heterodimer formation, prevention ofHER2 cleavage and others (Baselga and Albanell, 2001). U.S. Pat. Nos.5,677,171; 5,772,997; 6,165,464; and 6,399,063 disclose the anti-HER2invention but neither teach nor suggest that the antibody blocksligand-independent receptor activation.

Embodiments of the Invention

One aspect of the present invention is directed to neutralizingantibodies and more generally to a molecule that comprises theantigen-binding portion of an antibody which blocks ligand-dependentactivation and constitutive, ligand-independent activation of a receptorprotein tyrosine kinase. According to one embodiment the RPTK is afibroblast growth factor receptor. According to another embodiment theRPTK is FGFR3.

Another aspect of the present invention is directed to moleculescomprising an antigen binding domain which blocks ligand-dependentactivation of an FGFR. In one aspect the FGFR is FGFR3.

The molecule having the antigen-binding portion of an antibody accordingto the present invention can be used in a method for blocking theligand-dependent activation and/or ligand independent (constitutive)activation of FGFR3. Preferred embodiments of such antibodies/molecules,obtained from an antibody library designated as HUCAL® (HumanCombinatorial Antibody Library) clone, is presented in Table 1A with theunique V_(H)-CDR3 and V_(L)-CDR3 sequences given.

In addition to sequencing of the clones, a series of biochemical assayswere performed to determine affinity and specificity of the molecules tothe respective receptors.

TABLE 1A HuCAL ® - Clone VH-CDR3 Sequence VL-CDR3 Sequence FrameworkMSPRO2 DFLGYEFDY QSYDYSADY VH1B_L3 (SEQ ID NO: 8) (SEQ ID NO: 9) MSPRO11YYGSSLYHYV FGGFIDY QSHHFYE VH1B_L2 (SEQ ID NO: 10) (SEQ ID NO: 11)MSPRO12 YHSWYEMGYY GSTVGYMFDY QSYDFDFA VH2_L3 (SEQ ID NO: 12) (SEQ IDNO: 13) MSPRO21 DNWFKPFSDV QQYDSIPY VH1A_k4 (SEQ ID NO: 14) (SEQ ID NO:15) MSPRO24 VNHWTYTFDY QQMSNYPD VH1A_k3 (SEQ ID NO: 16) (SEQ ID NO: 17)MSPRO26 GYWYAYFTYI NYGYFDN QSYDNNSDV VH1B_L2 (SEQ ID NO: 18) (SEQ ID NO:19) MSPRO28 GGGWVSHGYY YLFDL FQYGSIPP VH1A_k1 (SEQ ID NO: 26) (SEQ IDNO: 27) MSPRO29 TWQYSYFYYL DGGYYFDI QQTNNAPV VH1B_k3 (SEQ ID NO: 20)(SEQ ID NO: 21) MSPRO54 NMAYTNYQYV NMPHFDY QSYDYFKL VH1B_L3 (SEQ ID NO:22) (SEQ ID NO: 23) MSPRO55 SMNSTMYWYL RRVLFDH QSYDMYMYI VH1B_L2 (SEQ IDNO: 28) (SEQ ID NO: 29) MSPRO59 SYYPDFDY QSYDGPDLW VH6_L3 (SEQ ID NO:24) (SEQ ID NO: 25) VH refers to the variable heavy chain, VL refers tothe variable light chain; L refers to the lambda light chain and krefers to the kappa light chain.

Table 1B lists the corresponding polynucleotide sequences of the CDRdomains.

TABLE 1B VL-CDR3 HuCAL ® VH-CDR3 polynucleotide polynucleotide Clonesequence sequence MSPRO2 GATTTTCTTG GTTATGAGTT CAGAGCTATG TGATTATACTATTCTGC TGATTAT (SEQ ID NO: 30) (SEQ ID NO: 31) MSPRO11 TATTATGGTTCTTCTCTTTA CAGTCTCATC TCATTATGTT TTTGGTGGTT ATTTTTATGA G TTATTGATTA T(SEQ ID NO: 33) (SEQ ID NO: 32) MSPRO12 TATCATTCTT GGTATGAGAT CAGAGCTATGGGGTTATTAT GGTTCTACTG ACTTTGATTT TGCT TTGGTTATAT GTTTGATTAT (SEQ ID NO:35) (SEQ ID NO: 34) MSPRO21 GATAATTGGT TTAAGCCTTT CAGCAGTATGATTTCTGATGTT TCTATTCCT TAT (SEQ ID NO: 36) (SEQ ID NO: 37) MSPRO24GTTAATCATT GGACTTATAC CAGCAGATGT TTTTGATTAT CTAATTATCC TGAT (SEQ ID NO:38) (SEQ ID NO: 39) MSPRO26 GGTTATTGGT ATGCTTATTT CAGAGCTATG TACTTATATTAATTATGGTT ACAATAATTC TGATGTT ATTTTGATAA T (SEQ ID NO: 41) (SEQ ID NO:40) MSPRO28 GGTGGTGGTT GGGTTTCTCA TTTCAGTATG TGGTTATTAT TATCTTTTTGGTTCTATTCC TCCT ATCTT (SEQ ID NO: 43) (SEQ ID NO: 42) MSPRO29 ACTTGGCAGTATTCTTATTT CAGCAGACTA TTATTATCTT GATGGTGGTT ATAATGCTCC TGTT ATTATTTTGATATT (SEQ ID NO: 45) (SEQ ID NO: 44) MSPRO54 AATATGGCTT ATACTAATTACAGAGCTATG TCAGTATGTT AATATGCCTC ACTATTTTAA GCTT ATTTTGATTA T (SEQ IDNO: 47) (SEQ ID NO: 46) MSPRO55 TCTATGAATT CTACTATGTA CAGAGCTATGACTTGGTAT CTTCGTCGTG ATGTATAATTAT ATT TTCTTTTTGA TCAT (SEQ ID NO: 49) (SEQID NO: 48) MSPRO59 TCTTATTATC CTGATTTTGA CAGAGCTATGAC TTAT GGTCCTGATCTTTGG (SEQ ID NO: 50) (SEQ ID NO: 51)

The polypeptide sequence of the VH and VL domains of the currentlypreferred embodiments of the present invention are presented below. FIG.30 provides the polynucleotide sequences of the preferred embodiments ofthe invention.

MS-Pro-2-VL (SEQ ID NO: 85) 1 DIELTQPPSV SVAPGQTARI SCSGDALGDKYASWYQQKPG QAPVLVIYDD 51 SDRPSGIPER FSGSNSGNTA TLTISGTQAE DEADYYCQSYDYSADYVFGG 101 GTKLTVLGQcorresponding to polynucleotide sequence having SEQ ID NO:67

MS-Pro-11-VL (SEQ ID NO: 86) 1 DIALTQPASV SGSPGQSITI SCTGTSSDVGGYNYVSWYQQ HPGKAPKLMI 51 YDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCQSHHFYEVFG 101 GGTKLTVLGQcorresponding to polynucleotide sequence having SEQ ID NO:63

MS-PRO-12-VL (SEQ ID NO: 87) 1 DIELTQPPSV SVAPGQTARI SCSGDALGDKYASWYQQKPG QAPVLVIYDD 51 SDRPSGIPER FSGSNSGNTA TLTISGTQAE DEADYYCQSYDFDFAVFGGG 101 TKLTVLGQcorresponding to polynucleotide sequence having SEQ ID NO:68

MS-Pro-21-VL (SEQ ID NO: 88) 1 DIVMTQSPDS LAVSLGERAT INCRSSQSVLYSSNNKNYLA WYQQKPGQPP 51 KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVAVYYCQQYDSI 101 PYTFGQGTKV EIKRTcorresponding to polynucleotide sequence having SEQ ID NO:60

MS-Pro-24-VL (SEQ ID NO: 89) 1 DIVLTQSPAT LSLSPGERAT LSCRASQSVSSSYLAWYQQK PGQAPRLLIY 51 GASSRATGVP ARFSGSGSGT DFTLTISSLE PEDFATYYCQQMSNYPDTFG 101 QGTKVEIKRTcorresponding to polynucleotide sequence having SEQ ID NO:57

MS-Pro-26-VL (SEQ ID NO: 90) 1 DIALTQPASV SGSPGQSITI SCTGTSSDVGGYNYVSWYQQ HPGKAPKLMI 51 YDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCQSYDNNSDVV 101 FGGGTKLTVL GQcorresponding to polynucleotide sequence having SEQ ID NO:64

MS-Pro-28-VL (SEQ ID NO: 91) 1 DIQMTQSPSS LSASVGDRVT ITCRASQGISSYLAWYQQKP GKAPKLLIYA 51 ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFAVYYCFQYGSIPPTFGQ 101 GTKVEIKRTcorresponding to polynucleotide sequence having SEQ ID NO:55

MS-Pro-29-VL (SEQ ID NO: 92) 1 DIVLTQSPAT LSLSPGERAT LSCRASQSVSSSYLAWYQQK PGQAPRLLIY 51 GASSRATGVP ARFSGSGSGT DFTLTISSLE PEDFATYYCQQTNNAPVTFG 101 QGTKVEIKRTcorresponding to polynucleotide sequence having SEQ ID NO:58

MS-Pro-54-VL (SEQ ID NO: 93) 1 DIELTQPPSV SVAPGQTARI SCSGDALGDKYASWYQQKPG QAPVLVIYDD 51 SDRPSGIPER FSGSNSGNTA TLTISGTQAE DEADYYCQSYDYFKLVFGGG 101 TKLTVLGQcorresponding to polynucleotide sequence having SEQ ID NO:66

MS-Pro-55-VL (SEQ ID NO: 94) 1 DIALTQPASV SGSPGQSITI SCTGTSSDVGGYNYVSWYQQ HPGKAPKLMI 51 YDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCQSYDMYNYIV 101 FGGGTKLTVL GQcorresponding to polynucleotide sequence having SEQ ID NO:62

MS-Pro-59-VL (SEQ ID NO: 95) 1 DIELTQPPSV SVAPGQTARI SCSGDALGDKYASWYQQKPG QAPVLVIYDD 51 SDRPSGIPER FSGSNSGNTA TLTISGTQAE DEADYYCQSYDGPDLWVFGG 101 GTKLTVLGQcorresponding to polynucleotide sequence having SEQ ID NO:69

MS-Pro-2-VH (SEQ ID NO: 96) 1 QVQLVQSGAE VKKPGASVKV SCKASGYTFTSYYMHWVRQA PGQGLEWMGW 51 INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSSLRSEDTAVYYCARDF 101 LGYEFDYWGQ GTLVTVSScorresponding to polynucleotide sequence having SEQ ID NO:77

MS-Pro-11-VH (SEQ ID NO: 97) 1 QVQLVQSGAE VKKPGASVKV SCKASGYTFTSYYMHWVRQA PGQGLEWMGW 51 INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSSLRSEDTAVYYCARYY 101 GSSLYHYVFG GFIDYWGQGT LVTVSScorresponding to polynucleotide sequence having SEQ ID NO:78

MS-Pro-12-VH (SEQ ID NO: 98) 1 QVQLKESGPA LVKPTQTLTL TCTFSGFSLSTSGVGVGWIR QPPGKALEWL 51 ALIDWDDDKY YSTSLKTRLT ISKDTSKNQV VLTMTNMDPVDTATYYCARY 101 HSWYEMGYYG STVGYMFDYW GQGTLVTVSScorresponding to polynucleotide sequence having SEQ ID NO:82

MS-Pro-21-VH (SEQ ID NO: 99) 1 QVQLVQSGAE VKKPGSSVKV SCKASGGTFSSYAISWVRQA PGQGLEWMGG 51 IIPIFGTANY AQKFQGRVTI TADESTSTAY MELSSLRSEDTAVYYCARDN 101 WFKPFSDVWG QGTLVTVSScorresponding to polynucleotide sequence having SEQ ID NO:71

MS-Pro-24-VH (SEQ ID NO: 100) 1 QVQLVQSGAE VKKPGSSVKV SCKASGGTFSSYAISWVRQA PGQGLEWMGG 51 IIPIFGTANY AQKFQGRVTI TADESTSTAY MELSSLRSEDTAVYYCARVN 101 HWTYTFDYWG QGTLVTVSScorresponding to polynucleotide sequence having SEQ ID NO:72

MS-Pro-26-VH (SEQ ID NO: 101) 1 QVQLVQSGAE VKKPGASVKV SCKASGYTFTSYYMHWVRQA PGQGLEWMGW 51 INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSSLRSEDTAVYYCARGY 101 WYAYFTYINY GYFDNWGQGT LVTVSScorresponding to polynucleotide sequence having SEQ ID NO:79

MS-Pro-28-VH (SEQ ID NO: 102) 1 QVQLVQSGAE VKKPGSSVKV SCKASGGTFSSYAISWVRQA PGQGLEWMGG 51 IIPIFGTANY AQKFQGRVTI TADESTSTAY MELSSLRSEDTAVYYCARGG 101 GWVSHGYYYL FDLWGQGTLV TVSScorresponding to polynucleotide sequence having SEQ ID NO:73

MS-Pro-29-VH (SEQ ID NO: 103) 1 QVQLVQSGAE VKKPGASVKV SCKASGYTFTSYYMHWVRQA PGQGLEWMGW 51 INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSSLRSEDTAVYYCARTW 101 QYSYFYYLDG GYYFDIWGQG TLVTVSScorresponding to polynucleotide sequence having SEQ ID NO:80

MS-Pro-54-VH (SEQ ID NO: 104) 1 QVQLVQSGAE VKKPGASVKV SCKASGYTFTSYYMHWVRQA PGQGLEWMGW 51 INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSSLRSEDTAVYYCARNM 101 AYTNYQYVNM PHFDYWGQGT LVTVSScorresponding to polynucleotide sequence having SEQ ID NO:75

MS-Pro-55-VH (SEQ ID NO: 105) 1 QVQLVQSGAE VKKPGASVKV SCKASGYTFTSYYMHWVRQA PGQGLEWMGW 51 INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSSLRSEDTAVYYCARSM 101 NSTMYWYLRR VLFDHWGQGT LVTVSScorresponding to polynucleotide sequence having SEQ ID NO:76

MS-Pro-59-VH (SEQ ID NO: 106) 1 QVQLQQSGPG LVKPSQTLSL TCAISGDSVSSNSAAWNWIR QSPGRGLEWL 51 GRTYYRSKWY NDYAVSVKSR ITINPDTSKN QFSLQLNSVTPEDTAVYYCA 101 RSYYPDFDYW GQGTLVTVSScorresponding to polynucleotide sequence having SEQ ID NO:84

In addition to sequencing of the clones, a series of biochemical assayswere performed to determine affinity and specificity of the molecules tothe respective receptors. Table 1C lists the affinity of the respectivemolecules to FGFR3 and FGFR1 as measured by BIACORE®-and/or FACS. In abinding assay to FGFR3-expressing cells, the IC₅₀ of the molecules wascalculated (Example 6). Domain specificity was determined as describedin Example 8. The ligand-independent inhibition of FGFR3 (neutralizingactivity) was determined as described in Example 10. Finally, themolecules were synthesized in a number of different formats includingFab, miniantibody (Fab-dHLX), IgG1, IgG4, IgG3 and as single chain Fv(scFv).

TABLE 1C Affinity Affinity IC₅₀ Ligand to FGFR3 to FGFR3 Affinity FR3independent BIAcore (FACS) to FGFR1 (FGF9 Domain inhibition AvailableClone nM nM nM Koff (s⁻¹) nM) Specificity of FGFR3 formats MSPRO59 1.5<1 — 7.1 × 10e−4 19 2 + Fab, Fab-dHLX IgG1, IgG4, mIgG3, scFv MSPRO2 3743 —   2 × 10e−2 360 2 ~ Fab, Fab-dHLX, IgG1, IgG4, MSPRO12 14 6.5 — 2.3× 10e−3 58 2 + Fab, Fab-dHLX, IgG1, IgG4, scFv MSPRO11 4 4 108   4 ×10e−4 220 3 Fab, Fab-dHLX MSPRO21 9 1.1 — 3.6 × 10e−3 50 3c Fab,Fab-dHLX MSPRO24 10 — 5.4 × 10e−3 70 3c Fab, IgG1 MSPRO26 4 1.4 32   5 ×10e−4 70 3 Fab, Fab-dHLX MSPRO28 9 0.3 160   4 × 10e−3 50 3 Fab MSPRO296 <1 29 1.4 × 10e−3 20 3c − Fab, IgG1, IgG4, Fab-dHLX, scFv MSPRO54 3.7NA 2.5   2 × 10e−3 45 3c Fab, IgG MSPRO55 2.9 NA — 7.4 × 10e−4 34 3c FabKey: affinity (as measured in nM) of the respective molecules to FGFR3and FGFR1 was measured by BIAcore ® and/or FACS. IC₅₀ were determinedfor the dimeric dHLX format of certain molecules having an antigenbinding site in an FDCP-FGFR3 proliferation assay performed with FGF9.Fab-dHLX refers to a Fab mini-antibody format where a dimer of the Fabmonomer is produced as a fusion protein after insertion into anexpression vector.

BIACORE® results for certain molecules

The numbers in Table 1D represent the IC₅₀s of the dimeric dHLX formatof certain binders (molecule with antigen binding site) in theFDCP-FGFR3 proliferation assay performed with FGF9. The numbers inparentheses are the IC₅₀ of the monomeric Fabs in the same assay. Table1E presents the K_(D) value for certain MSPRO molecules in miniantibodyform, as determined in the BIACORE® assay.

TABLE 1D binder IC₅₀ MSPRO2 61 nM (360) MSPRO12 26 nM (58) MSPRO21 20 nM(50) MSPRO26 8 nM (70)

TABLE 1E K_(D) determination for certain molecules BIAcore Number ofClone K_(D) [nM] measurements MS-Pro-2-dHLX-MH 4.3 (37) 1MS-Pro-11-dHLX-MH 0.7 (4)  1 MS-Pro-12-dHLX-MH 1.2 (14) 1MS-Pro-21-dHLX-MH  2.2 (4.1) 1 MS-Pro-24-dHLX-MH   2 (10) 1MS-Pro-26-dHLX-MH  2 (4) 1 MS-Pro-28-dHLX-MH 1.6 (9)  1

Certain non-limiting embodiments of molecules according to the presentinvention that block constitutive (ligand-independent) activation ofFGFR3 are referred to herein MSPRO2, MSPRO12 and MSPRO59 comprisingV_(H)-CDR3 and V_(L)-CDR3 domains having SEQ ID NO:8 and SEQ ID NO:9;SEQ ID NO:12 and SEQ ID NO:13; and SEQ ID NO:24 and SEQ ID NO:25,respectively. The preferred, but non-limiting, embodiments of moleculesaccording to the present invention that block ligand-dependentactivation of FGFR3 are referred to herein MSPRO11, MSPRO21, MSPRO24,MSPRO26, MSPRO29, and MSPRO54 comprising VH-CDR3 and VL-CDR3 domainshaving SEQ ID NO:10 and SEQ ID NO:11; SEQ ID NO:14 and SEQ ID NO:15; SEQID NO:16 and SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19; SEQ ID NO:21and SEQ ID NO:22; SEQ ID NO:23 and SEQ ID NO:24, respectively. Anantibody or a molecule of the present invention is said to haveincreased affinity for a RPTK if it binds a soluble dimeric form of saidRPTK with a K_(D) of less than about 50 nM, preferably less than about30 nM and more preferably less than about 10 nM, as determined by theBIACORE® chip assay for affinity, by a FACS-Scatchard analysis or othermethods known in the art.

For convenience, Tables 1F and 1G outline the pairs of moleculesaccording to their peptide SEQ ID NO and nucleotide SEQ ID NO,respectively.

TABLE 1F Peptide pairs Fragment V heavy chain V light chain antibody #CDR3 CDR3 V heavy chain V light chain MSPRO2 SEQ ID NO: 8 SEQ ID NO: 9SEQ ID NO: 96 SEQ ID NO: 85 MSPRO12 SEQ ID NO: 12 SEQ ID NO: 13 SEQ IDNO: 98 SEQ ID NO: 87 MSPRO59 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 106SEQ ID NO: 95 MSPRO11 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 97 SEQ IDNO: 86 MSPRO21 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 99 SEQ ID NO: 88MSPRO24 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 100 SEQ ID NO: 89 MSPRO26SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 101 SEQ ID NO: 90 MSPRO28 SEQ IDNO: 26 SEQ ID NO: 27 SEQ ID NO: 102 SEQ ID NO: 91 MSPRO29 SEQ ID NO: 20SEQ ID NO: 21 SEQ ID NO: 103 SEQ ID NO: 92 MSPRO54 SEQ ID NO: 22 SEQ IDNO: 23 SEQ ID NO: 104 SEQ ID NO: 93 MSPRO55 SEQ ID NO: 28 SEQ ID NO: 29SEQ ID NO: 105 SEQ ID NO: 94

TABLE 1G Nucleotide pairs fragment V heavy chain V light chain antibody# CDR3 CDR3 V heavy chain V light chain MSPRO2 SEQ ID NO: 30 SEQ ID NO:31 SEQ ID NO: 77 SEQ ID NO: 67 MSPRO12 SEQ ID NO: 34 SEQ ID NO: 35 SEQID NO: 82 SEQ ID NO: 68 MSPRO59 SEQ ID NO: 50 SEQ ID NO: 51 SEQ ID NO:84 SEQ ID NO: 69 MSPRO11 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 78 SEQID NO: 63 MSPRO21 SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 71 SEQ ID NO:60 MSPRO24 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 72 SEQ ID NO: 57MSPRO26 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 79 SEQ ID NO: 64 MSPRO28SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 73 SEQ ID NO: 55 MSPRO29 SEQ IDNO: 44 SEQ ID NO: 45 SEQ ID NO: 80 SEQ ID NO: 58 MSPRO54 SEQ ID NO: 46SEQ ID NO: 47 SEQ ID NO: 75 SEQ ID NO: 66 MSPRO55 SEQ ID NO: 48 SEQ IDNO: 49 SEQ ID NO: 76 SEQ ID NO: 62

While the specific discovery of an antibody/molecule that blocksconstitutive activation was made with respect to FGFR3 using a solubledimeric form of FGFR3 to screen a phage display antibody library, it isbelieved that for all, or almost all, receptor protein tyrosine kinases,antibodies/molecules that block constitutive activation can be similarlyobtained using a soluble dimeric form of a corresponding extracellulardomain of a receptor protein tyrosine kinase. Non-limiting examples ofreceptor protein tyrosine kinases disclosed in Blume-Jensen et al.(2001) include EGFR/ErbB1, ErbB2/HER2/Neu, ErbB/HER3, ErbB4/HER4,IGF-1R, PDGFR-a, PDGFR-β, CSF-1R, kit/SCFR, Flk2/FH3, Flk1/VEGFR1,Flk1/VEGFR2, Flt4/VEGFR3, FGFR1, FGFR2/K-SAM, FGFR3, FGFR4, TrkA, TrkC,HGFR, RON, EphA2, EphB2, EphB4, Axl, TIE/TIE1, Tek/TIE2, Ret, ROSAlk,Ryk, DDR, LTK and MUSK.

Furthermore, antibodies/molecules that block ligand-dependent orligand-independent activation of heterodimer receptor protein tyrosinekinases are intended to be included in the scope of the invention.Heterodimerization is well documented for members of the EGFR subfamilyof receptor protein tyrosine kinases and others. Non-limiting examplesinclude EGFR/PDGFRβ, Flt1/KDR and EGFR/ErbB2 heterodimers. EGFR andPDGFRβ heterodimers have been identified as a mechanism for PDGF signaltransduction in rat vascular smooth muscle cells (Saito et al., 2001)and Flt-1/KDR heterodimers are required for vinculin assembly in focaladhesions in response to VEGF signaling (Sato et al., 2000).

The present invention is also directed to a molecule having theantigen-binding portion of an antibody which binds to a dimeric form ofan extracellular portion of a receptor protein tyrosine kinase (RPTK),such as a FGFR, and blocks the ligand-independent (constitutive)activation and/or ligand-dependent activation of the RPTK.

Further provided is a method for screening a molecule comprising theantigen-binding portion of an antibody which blocks ligand-independentor ligand-dependent activation of a receptor protein tyrosine kinase,comprising:

-   -   providing a library of antigen binding fragments;    -   screening a library of antigen binding fragments for binding to        a dimeric form of a receptor protein tyrosine kinase;    -   identifying an antigen binding fragment which binds to the        dimeric form of the receptor protein tyrosine kinase as a        candidate molecule for blocking constitutive activation of the        receptor protein tyrosine kinase; and    -   determining whether the candidate molecule blocks constitutive        and or ligand-dependent activation of the receptor protein        tyrosine kinase in a cell.        Antibodies

Antibodies, or immunoglobulins, comprise two heavy chains linkedtogether by disulfide bonds and two light chains, each light chain beinglinked to a respective heavy chain by disulfide bonds in a “Y” shapedconfiguration. Proteolytic digestion of an antibody yields Fv (fragmentvariable comprising Fab domains) and Fc (fragment crystalline) domains.The antigen binding domains, Fab′, include regions where the polypeptidesequence varies. The term F(ab′)₂ represents two Fab′ arms linkedtogether by disulfide bonds. The central axis of the antibody is termedthe Fc fragment. Each heavy chain has at one end a variable domain(V_(H)) followed by a number of constant domains (C_(H)). Each lightchain has a variable domain (V_(L)) at one end and a constant domain(C_(L)) at its other end, the light chain variable domain being alignedwith the variable domain of the heavy chain and the light chain constantdomain being aligned with the first constant domain of the heavy chain(C_(H1)). The variable domains of each pair of light and heavy chainsform the antigen binding site. The domains on the light and heavy chainshave the same general structure and each domain comprises four frameworkregions, whose sequences are relatively conserved, joined by threehypervariable domains known as complementarity determining regions(CDR1-3). These domains contribute specificity and affinity of theantigen binding site. The isotype of the heavy chain (gamma, alpha,delta, epsilon or mu) determines immunoglobulin class (IgG, IgA, IgD,IgE or IgM, respectively). The light chain is either of two isotypes(kappa, ? or lambda, ?) found in all antibody classes.

It should be understood that when the terms “antibody” or “antibodies”are used, this is intended to include intact antibodies, such aspolyclonal antibodies or monoclonal antibodies (mAbs), as well asproteolytic fragments thereof such as the Fab or F(ab′)₂ fragments.Further included within the scope of the invention are chimericantibodies; human and humanized antibodies; recombinant and engineeredantibodies, and fragments thereof. Furthermore, the DNA encoding thevariable region of the antibody can be inserted into the DNA encodingother antibodies to produce chimeric antibodies (see, for example, U.S.Pat. No. 4,816,567). Single chain antibodies fall within the scope ofthe present invention. Single chain antibodies can be single chaincomposite polypeptides having antigen binding capabilities andcomprising amino acid sequences homologous or analogous to the variableregions of an immunoglobulin light and heavy chain (linked V_(H)-V_(L)or single chain Fv (scFv)). Both V_(H) and V_(L) may copy naturalmonoclonal antibody sequences or one or both of the chains may comprisea CDR-FR construct of the type described in U.S. Pat. No. 5,091,513, theentire contents of which are incorporated herein by reference. Theseparate polypeptides analogous to the variable regions of the light andheavy chains are held together by a polypeptide linker. Methods ofproduction of such single chain antibodies, particularly where the DNAencoding the polypeptide structures of the V_(H) and V_(L) chains areknown, may be accomplished in accordance with the methods described, forexample, in U.S. Pat. Nos. 4,946,778, 5,091,513 and 5,096,815, theentire contents of which are hereby incorporated by reference.

Additionally, CDR grafting may be performed to alter certain propertiesof the antibody molecule including affinity or specificity. Anon-limiting example of CDR grafting is disclosed in U.S. Pat. No.5,225,539.

A “molecule having the antigen-binding portion of an antibody” as usedherein is intended to include not only intact immunoglobulin moleculesof any isotype and generated by any animal cell line or microorganism,but also the antigen-binding reactive fraction thereof, including, butnot limited to, the Fab fragment, the Fab′ fragment, the F(ab′)₂fragment, the variable portion of the heavy and/or light chains thereof,Fab miniantibodies (see WO 93/15210, U.S. patent application Ser. No.08/256,790, WO 96/13583, U.S. patent application Ser. No. 08/817,788, WO96/37621, U.S. patent application Ser. No. 08/999,554, the entirecontents of which are incorporated herein by reference) and chimeric orsingle-chain antibodies incorporating such reactive fraction, as well asany other type of molecule or cell in which such antibody reactivefraction has been physically inserted, such as a chimeric T-cellreceptor or a T-cell having such a receptor, or molecules developed todeliver therapeutic moieties by means of a portion of the moleculecontaining such a reactive fraction. Such molecules may be provided byany known technique, including, but not limited to, enzymatic cleavage,peptide synthesis or recombinant techniques.

The term “Fc” as used herein is meant as that portion of animmunoglobulin molecule (Fragment crystallizable) that mediatesphagocytosis, triggers inflammation and targets Ig to particulartissues; the Fc portion is also important in complement activation.

In one embodiment of the invention, a chimera comprising a fusion of theextracellular domain of the RPTK and an immunoglobulin constant domaincan be constructed useful for assaying for ligands for the receptor andfor screening for antibodies and fragments thereof.

The “extracellular domain” when used herein refers the polypeptidesequence of the RPTKs disclosed herein which are normally positioned tothe outside of the cell. The extracellular domain encompassespolypeptide sequences in which part or all of the adjacent (C-terminal)hydrophobic transmembrane and intracellular sequences of the mature RPTKhave been deleted. Thus, the extracellular domain-containing polypeptidecan comprise the extracellular domain and a part of the transmembranedomain. Alternatively, in the preferred embodiment, the polypeptidecomprises only the extracellular domain of the RPTK. The truncatedextracellular domain is generally soluble. The skilled practitioner canreadily determine the extracellular and transmembrane domains of a RPTKby aligning the RPTK of interest with known RPTK amino acid sequencesfor which these domains have been delineated. Alternatively, thehydrophobic transmembrane domain can be readily delineated based on ahydrophobicity plot of the polypeptide sequence. The extracellulardomain is N-terminal to the transmembrane domain.

The term “epitope” is meant to refer to that portion of any moleculecapable of being bound by an antibody or a fragment thereof which canalso be recognized by that antibody. Epitopes or antigenic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

A “neutralizing antibody” as used herein refers to a molecule having theantigen binding site to a specific receptor capable of reducing orinhibiting (blocking) activity or signaling through a receptor, asdetermined by in vivo or in vitro assays, as per the specification.

A monoclonal antibody (mAb) is a substantially homogeneous population ofantibodies to a specific antigen. MAbs may be obtained by methods knownto those skilled in the art. See, for example Kohler et al (1975); U.S.Pat. No. 4,376,110; Ausubel et al (1987-1999); Harlow et al (1988); andColligan et al (1993), the contents of which references are incorporatedentirely herein by reference. The mAbs of the present invention may beof any immunoglobulin class including IgG, IgM, IgE, IgA, and anysubclass thereof. A hybridoma producing an mAb may be cultivated invitro or in vivo. High titers of mAbs can be obtained in in vivoproduction where cells from the individual hybridomas are injectedintraperitoneally into pristine-primed Balb/c mice to produce ascitesfluid containing high concentrations of the desired mAbs. MAbs ofisotype IgM or IgG may be purified from such ascites fluids, or fromculture supernatants, using column chromatography methods well known tothose of skill in the art.

Chimeric antibodies are molecules, the different portions of which arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Antibodies which have variable region framework residuessubstantially from human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse antibody(termed a donor antibody) are also referred to as humanized antibodies.Chimeric antibodies are primarily used to reduce immunogenicity inapplication and to increase yields in production, for example, wheremurine mAbs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric mAbs are used. Chimericantibodies and methods for their production are known in the art (Betteret al, 1988; Cabilly et al, 1984; Harlow et al, 1988; Liu et al, 1987;Morrison et al, 1984; Boulianne et al, 1984; Neuberger et al, 1985;Sahagan et al , 1986; Sun et al, 1987; Cabilly et al; European PatentApplications 125023, 171496, 173494, 184187, 173494, PCT patentapplications WO 86/01533, WO 97/02671, WO 90/07861, WO 92/22653 and U.S.Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539).These references are hereby incorporated by reference.

Besides the conventional method of raising antibodies in vivo,antibodies can be generated in vitro using phage display technology.Such a production of recombinant antibodies is much faster compared toconventional antibody production and they can be generated against anenormous number of antigens. In contrast, in the conventional method,many antigens prove to be non-immunogenic or extremely toxic, andtherefore cannot be used to generate antibodies in animals. Moreover,affinity maturation (i.e., increasing the affinity and specificity) ofrecombinant antibodies is very simple and relatively fast. Finally,large numbers of different antibodies against a specific antigen can begenerated in one selection procedure. To generate recombinant monoclonalantibodies one can use various methods all based on phage displaylibraries to generate a large pool of antibodies with different antigenrecognition sites. Such a library can be made in several ways: One cangenerate a synthetic repertoire by cloning synthetic CDR3 regions in apool of heavy chain germline genes and thus generating a large antibodyrepertoire, from which recombinant antibody fragments with variousspecificities can be selected. One can use the lymphocyte pool of humansas starting material for the construction of an antibody library. It ispossible to construct naive repertoires of human IgM antibodies and thuscreate a human library of large diversity. This method has been widelyused successfully to select a large number of antibodies againstdifferent antigens. Protocols for bacteriophage library construction andselection of recombinant antibodies are provided in the well-knownreference text, Current Protocols in Immunology, Colligan et al (Eds.),John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

Another aspect of the present invention is directed to a method forscreening for the antibody or molecule of the present invention byscreening a library of antibody fragments displayed on the surface ofbacteriophage, such as disclosed in the Examples herein and described inWO 97/08320, U.S. Pat. No. 6,300,064 and Knappik et al. (2000), forbinding to a soluble dimeric form of a receptor protein tyrosine kinase.An antibody fragment which binds to the soluble dimeric form of the RPTKused for screening is identified as a candidate molecule for blockingligand-dependent activation and/or constitutive activation of the RPTKin a cell. Preferably the RPTK of which a soluble dimeric form is usedin the screening method is a fibroblast growth factor receptor (FGFR),and most preferably FGFR3.

As a first screening method, the soluble dimeric form of a receptortyrosine kinase can be constructed and prepared in a number of differentways. For instance, the extracellular domain of a RPTK joined to Fc andexpressed as a fusion polypeptide that dimerizes naturally by means ofthe Fc portion of the RPTK-Fc fusion. Other suitable types of constructsof FGFR3, serving as guidance for other RPTKs, are disclosed in theExamples presented herein.

The assays for determining binding of antibody fragments to FGFR3,binding affinities, inhibition of cell proliferation, etc., are alsodescribed in the Examples herein below.

The term “cell proliferation” refers to the rate at which a group ofcells divides. The number of cells growing in a vessel can be quantifiedby a person skilled in the art when that person visually counts thenumber of cells in a defined volume using a common light microscope.Alternatively, cell proliferation rates can be quantified by laboratoryapparati that optically or conductively measure the density of cells inan appropriate medium.

A second screen for antibody fragments as candidate molecules can bedone using cells having very high overexpression of the RPTK, such asfor instance RCJ-M15 cells overexpressing mutant (achondroplasia) FGFR3.In cells expressing very high levels of receptor some ligand-independentactivation occurs even without the presence of a mutation, such as aconstitutive activation mutation. It is believed that RPTKoverexpression forces RPTKs to dimerize and signal even in the absenceof ligand. These cells have monomeric receptors as well as dimericreceptors present on their cell surface. Using this type of cell, one ofskill in the art would be able to identify all different kinds ofantibodies, i.e., blocking ligand-dependent activation, blockingconstitutive activation, blocking activation and binding only tomonomeric form, etc.

Other screens can be carried out on cell lines expressing a RPTKcarrying a mutation, such as the FDCP-FR3ach line expressing the FGFR3achondroplasia mutation. The receptors of this line becomeconstitutively active, e.g. are able to signal in the absence of aligand as determined by ERK (MAPK) phosphorylation, a downstreameffector.

A further aspect of the present invention relates to a method fortreating or inhibiting a skeletal dysplasia or craniosynostosis disorderassociated with constitutive activation of a RPTK which involvesadministering the molecule of the present invention to a subject needthereof. Non-limiting examples of skeletal dysplasias includeachondroplasia, thanatophoric dysplasia (TDI or TDII),hypochondroplasia, and severe achondroplasia with developmental delayand acanthosis nigricans (SADDAN) dysplasia. Non-limiting examples ofcraniosynostosis disorder are Muenke coronal craniosynostosis andCrouzon syndrome with acanthosis nigricans. The symptoms and etiology ofthese diseases and disorders are reviewed in Vajo et al. (Vajo et al,2000).

The present invention also provides for a method for treating orinhibiting a cell proliferative disease or disorder associated with theaction of an abnormal constitutively activated RPTK, for example tumorformation, primary tumors, tumor progression or tumor metastasis. Amolecule comprising at least one antigen binding portion of an antibodythat blocks constitutive activation of a RPTK is administered to asubject in need thereof to treat or inhibit such a cell proliferativedisease or disorder.

The terms “treating or inhibiting a proliferative disease or disorder”or “treating or inhibiting a tumor” are used herein and in the claims toencompass tumor formation, primary tumors, tumor progression or tumormetastasis.

Tumor formation or tumor growth are intended to encompass solid andnon-solid tumors. Solid tumors include mammary, ovarian, prostate,colon, cervical, gastric, esophageal, papillary thyroid, pancreatic,bladder, colorectal, melanoma, small-cell lung and non-small-cell lungcancers, granulose cell carcinoma, transitional cell carcinoma, vasculartumors, all types of sarcomas, e.g. osteosarcoma, chondrosarcoma,Kaposi's sarcoma, myosarcoma, hemangiosarcoma, and glioblastomas.

Non-solid tumors include for example hematopoietic malignancies such asall types of leukemia, e.g. chronic myelogenous leukemia (CML), acutemyelogenous leukemia (AML), mast cell leukemia, chronic lymphocyticleukemia (CLL) and acute lymphocytic leukemia (ALL), lymphomas, andmultiple myeloma (MM). FGFR3 has been implicated in poor prognosis seenin some patients.

Tumor progression is the phenomenon whereby cancers become moreaggressive with time. Progression can occur in the course of continuousgrowth, or when a tumor recurs after treatment and includes progressionof transitional cell carcinoma, osteo or chondrosarcoma, multiplemyeloma, and mammary carcinoma (one of the known RPTKs involved inmammary carcinoma is ErbB3).

The role of the FGFR3 in tumor progression associated with transitionalcell carcinoma and multiple myeloma has recently been elucidated(Cappellen, et al, 1999; Chesi, et al, 2001)

In another aspect of the present invention, molecules which bind FGFR,more preferably FGFR3, and block ligand-dependent receptor activationare provided. These molecules are useful in treating hyperproliferativediseases or disorders and non-neoplastic angiogenic pathologicconditions such as neovascular glaucoma, proliferative retinopathyincluding proliferative diabetic retinopathy, macular degeneration,hemangiomas, angiofibromas, and psoriasis. The role of FGFs and theirreceptors in neo- and hypervascularization has been well documented(Frank, 1997; Gerwins et al, 2000)

In another aspect of the present invention, the pharmaceuticalcompositions according to the present invention is similar to those usedfor passive immunization of humans with other antibodies. Typically, themolecules of the present invention comprising the antigen bindingportion of an antibody will be suspended in a sterile saline solutionfor therapeutic uses. The pharmaceutical compositions may alternativelybe formulated to control release of active ingredient (moleculecomprising the antigen binding portion of an antibody) or to prolong itspresence in a patient's system. Numerous suitable drug delivery systemsare known and include, e.g., implantable drug release systems,hydrogels, hydroxymethylcellulose, microcapsules, liposomes,microemulsions, microspheres, and the like. Controlled releasepreparations can be prepared through the use of polymers to complex oradsorb the molecule according to the present invention. For example,biocompatible polymers include matrices of poly(ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebaric acid (Sherwood et al, 1992). The rate of release ofthe molecule according to the present invention, i.e., of an antibody orantibody fragment, from such a matrix depends upon the molecular weightof the molecule, the amount of the molecule within the matrix, and thesize of dispersed particles (Saltzman et al., 1989 and Sherwood et al.,1992). Other solid dosage forms are described in (Ansel et al., 1990 andGennaro, 1990).

The pharmaceutical composition of this invention may be administered byany suitable means, such as orally, intranasally, subcutaneously,intramuscularly, intravenously, intra-arterially, intralesionally orparenterally. Ordinarily, intravenous (i.v.) or parenteraladministration will be preferred.

It will be apparent to those of ordinary skill in the art that thetherapeutically effective amount of the molecule according to thepresent invention will depend, inter alia, upon the administrationschedule, the unit dose of molecule administered, whether the moleculeis administered in combination with other therapeutic agents, the immunestatus and health of the patient, the therapeutic activity of themolecule administered and the judgment of the treating physician. Asused herein, a “therapeutically effective amount” refers to the amountof a molecule required to alleviate one or more symptoms associated witha disorder being treated over a period of time.

Although an appropriate dosage of a molecule of the invention variesdepending on the administration route, age, body weight, sex, orconditions of the patient and should be ultimately determined by thephysician, in the case of oral administration, the daily dosage cangenerally be between about 0.01-200 mg, preferably about 0.01-10 mg,more preferably about 0.1-10 mg, per kg body weight. In the case ofparenteral administration, the daily dosage can generally be betweenabout 0.001-100 mg, preferably about 0.001-1 mg, more preferably about0.01-1 mg, per kg body weight. The daily dosage can be administered, forexample, in regimens typical of 1-4 individual administrations daily.Various considerations in arriving at an effective amount are described,e.g., in Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, 8th ed., Pergamon Press, 1990; and Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa.,1990.

The molecule of the present invention as an active ingredient isdissolved, dispersed or admixed in an excipient that is pharmaceuticallyacceptable and compatible with the active ingredient as is well known.Suitable excipients are, for example, water, saline, phosphate bufferedsaline (PBS), dextrose, glycerol, ethanol, or the like and combinationsthereof. Other suitable carriers are well-known to those in the art.(See, for example, Ansel et al., 1990 and Gennaro, 1990). In addition,if desired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, or pH bufferingagents.

Combination Therapy

The combined treatment of one or more of the molecules of the inventionwith an anti-neoplastic or anti-chemotherapeutic drug such asdoxorubicin, cisplatin or TAXOL® provides a more efficient treatment forinhibiting the growth of tumor cells than the use of the molecule byitself. In one embodiment, the pharmaceutical composition comprises theantibody and carrier with an anti-chemotherapeutic drug.

The present invention also provides for an isolated acid molecule, whichcomprises a polynucleotide sequence encoding the molecule having atleast one antigen binding portion of an antibody that blocksligand-dependent activation and/or constitutive activation of a receptorprotein tyrosine kinase such as FGFR3, and a host cell comprising thisnucleic acid molecule. Furthermore, also within the scope of the presentinvention is a nucleic acid molecule containing a polynucleotidesequence having at least 90% sequence identity, preferably about 95%,and more preferably about 97% identity to the above encoding nucleotidesequence as would well understood by those of skill in the art.

The invention also provides isolated nucleic acid molecule thathybridizes under high stringency conditions to polynucleotides havingSEQ ID NO:30 through SEQ ID NO:51 and SEQ ID NOS: 55, 57-58, 60, 62-64,66-69, 71-73, 75-80, 82, 84 or the complement thereof. As used herein,highly stringent conditions are those which are tolerant of up to about5-20% sequence divergence, preferably about 5-10%. Without limitation,examples of highly stringent (−10° C. below the calculated Tm of thehybrid) conditions use a wash solution of 0.1.times.SSC (standard salinecitrate) and 0.5% SDS at the appropriate Ti below the calculated Tm ofthe hybrid. The ultimate stringency of the conditions is primarily dueto the washing conditions, particularly if the hybridization conditionsused are those which allow less stable hybrids to form along with stablehybrids. The wash conditions at higher stringency then remove the lessstable hybrids. A common hybridization condition that can be used withthe highly stringent to moderately stringent wash conditions describedabove is hybridization in a solution of 6×SSC (or 6×SSPE), 5× Denhardt'sreagent, 0.5% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA atan appropriate incubation temperature Ti. See generally Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring HarborPress (1989)) for suitable high stringency conditions.

Stringency conditions are a function of the temperature used in thehybridization experiment and washes, the molarity of the monovalentcations in the hybridization solution and in the wash solution(s) andthe percentage of formamide in the hybridization solution. In general,sensitivity by hybridization with a probe is affected by the amount andspecific activity of the probe, the amount of the target nucleic acid,the detectability of the label, the rate of hybridization, and theduration of the hybridization. The hybridization rate is maximized at aTi (incubation temperature) of 20-25° C. below Tm for DNA:DNA hybridsand 10-15° C. below Tm for DNA:RNA hybrids. It is also maximized by anionic strength of about 1.5M Na⁺. The rate is directly proportional toduplex length and inversely proportional to the degree of mismatching.

Specificity in hybridization, however, is a function of the differencein stability between the desired hybrid and “background” hybrids. Hybridstability is a function of duplex length, base composition, ionicstrength, mismatching, and destabilizing agents (if any).

The Tm of a perfect hybrid may be estimated for DNA:DNA hybrids usingthe equation of Meinkoth et al (1984), asTm=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/Land for DNA:RNA hybrids, asTm=79.8° C.+18.5(log M)+0.58(% GC)−11.8(% GC)²−0.56(% form)−820/Lwhere

-   -   M, molarity of monovalent cations, 0.01-0.4 M NaCl,    -   % GC, percentage of G and C nucleotides in DNA, 30%-75%,    -   % form, percentage formamide in hybridization solution, and    -   L, length hybrid in base pairs.

Tm is reduced by 0.5-1.5° C. (an average of 1° C. can be used for easeof calculation) for each 1% mismatching.

The Tm may also be determined experimentally. As increasing length ofthe hybrid (L) in the above equations increases the Tm and enhancesstability, the full-length rat gene sequence can be used as the probe.

Filter hybridization is typically carried out at 68° C., and at highionic strength (e.g., 5-6×SSC), which is non-stringent, and followed byone or more washes of increasing stringency, the last one being of theultimately desired high stringency. The equations for Tm can be used toestimate the appropriate Ti for the final wash, or the Tm of the perfectduplex can be determined experimentally and Ti then adjustedaccordingly.

The present invention also relates to a vector comprising the nucleicacid molecule of the present invention. The vector of the presentinvention may be, for example, a plasmid, cosmid, virus, bacteriophageor another vector used conventionally in genetic engineering, and maycomprise further genes such as marker genes which allow for theselection of said vector in a suitable host cell and under suitableconditions.

Furthermore, the vector of the present invention may, in addition to thenucleic acid sequences of the invention, comprise expression controlelements, allowing proper expression of the coding regions in suitablehosts. Such control elements are known to the artisan and may include apromoter, a splice cassette, translation initiation codon, translationand insertion site for introducing an insert into the vector.

Preferably, the nucleic acid molecule of the invention is operativelylinked to said expression control sequences allowing expression ineukaryotic or prokaryotic cells.

Control elements ensuring expression in eukaryotic or prokaryotic cellsare well known to those skilled in the art. As mentioned herein above,they usually comprise regulatory sequences ensuring initiation oftranscription and optionally poly-A signals ensuring termination oftranscription and stabilization of the transcript.

Methods for construction of nucleic acid molecules according to thepresent invention, for construction of vectors comprising said nucleicacid molecules, for introduction of said vectors into appropriatelychosen host cells, for causing or achieving the expression arewell-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al.,1994).

The invention also provides for conservative amino acid variants of themolecules of the invention. Variants according to the invention also maybe made that conserve the overall molecular structure of the encodedproteins. Given the properties of the individual amino acids comprisingthe disclosed protein products, some rational substitutions will berecognized by the skilled worker. Amino acid substitutions, i.e.“conservative substitutions,” may be made, for instance, on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.

For example: (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt a-helices. Similarly, certainamino acids, such as alanine, cysteine, leucine, methionine, glutamicacid, glutamine, histidine and lysine are more commonly found ina-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophanand threonine are more commonly found in β-pleated sheets. Glycine,serine, aspartic acid, asparagine, and proline are commonly found inturns. Some preferred substitutions may be made among the followinggroups: (i) S and T; (ii) P and G; and (iii) A, V, L and 1. Given theknown genetic code, and recombinant and synthetic DNA techniques, theskilled scientist readily can construct DNAs encoding the conservativeamino acid variants.

As used herein, “sequence identity” between two polypeptide sequencesindicates the percentage of amino acids that are identical between thesequences. “Sequence similarity” indicates the percentage of amino acidsthat either are identical or that represent conservative amino acidsubstitutions.

Conjugates

One embodiment of the present invention provides molecules of thepresent invention conjugated to cytotoxins. The cytotoxic moiety of theantibody may be a cytotoxic drug or an enzymatically active toxin orbacterial or plant origin, or an enzymatically active fragment of such atoxin including, but not limited to, diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, curcin,crotin, saponin, gelonin, mitogellin, restrictocin, phenomycin, andenomycin. In another embodiment, the molecules of the present inventionare conjugated to small molecule anti-cancer drugs. Conjugates of theantibody and such cytotoxic moieties are made using a variety ofbifunctional protein coupling agents. Examples of such reagents includeSPDP, IT, bifunctional derivatives of imidoesters such a dimethyladipimidate HCl, active esters such as disuccinimidyl suberate,aldehydes such as glutaraldehyde, bis-azido compounds such asbis-(p-azidobenzoyl) hexanediamine, bis-diazonium derivatives,dissocyanates and bis-active fluorine compounds. The lysing portion of atoxin may be joined to the Fab fragment of the antibodies.

Additionally, the molecules of the present invention can also bedetected in vivo by imaging, for example imaging of cells which haveundergone tumor progression or have metastasized. Antibody labels ormarkers for in vivo imaging of RPTKs include those detectable byX-radiography, NMR, PET, or ESR. For X-radiography, suitable labelsinclude radioisotopes such as barium or cesium, which emit detectableradiation but are not overtly harmful to the subject. Suitable markersfor NMR and ESR include those with a detectable characteristic spin,such as deuterium, which may be incorporated into the antibody.

A specific antibody or antibody portion which has been labeled with anappropriate detectable imaging moiety, such as a radioisotope (forexample, ¹³¹I, ¹¹¹In, ⁹⁹Tc), a radio-opaque substance, or a materialdetectable by nuclear magnetic resonance, is introduced (for example,parenterally, subcutaneously or intraperitoneally) into the mammal to beexamined for a disorder. It will be understood in the art that the sizeof the subject and the imaging system used will determine the quantityof imaging moieties needed to produce diagnostic images. In the case ofa radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries. The labeled antibody or antibody portion will thenpreferentially accumulate at the location of cells which contain aspecific RPTK. In vivo tumor imaging is described in Burchiel et al.,(1982).

The methods and compositions described herein may be performed, forexample, by utilizing pre-packaged diagnostic test kits comprising inone or more containers (i) at least one immunoglobulin of the inventionand (ii) a reagent suitable for detecting the presence of saidimmunoglobulin when bound to its target. A kit may be conveniently used,e.g., in clinical settings or in home settings, to diagnose patientsexhibiting a disease (e.g., skeletal dysplasia, craniosynostosisdisorders, cell proliferative diseases or disorders, or tumorprogression), and to screen and identify those individuals exhibiting apredisposition to such disorders. A composition of the invention alsomay be used in conjunction with a reagent suitable for detecting thepresence of said immunoglobulin when bound to its target, as well asinstructions for use, to carry out one or more methods of the invention.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention.

EXAMPLES

An important approach to controlling cellular FGFR3 activity is thegeneration of reagents that block receptor signaling. Without wishing tobe bound by theory, molecules which bind the extracellular domain of thereceptor may inhibit the receptor by competing with FGF or heparinbinding or, alternatively, by preventing receptor dimerization.Additionally, binding to the extracellular domain may acceleratereceptor internalization and turnover. Humanized antibodies are expectedto have inhibitory/neutralizing action and are of particular interestsince they are considered to be valuable for therapeutic applications,especially by avoiding the human anti-mouse antibody response frequentlyobserved with rodent antibodies. The experiments in which theneutralizing antibodies are screened, identified and obtained usingfully synthetic human antibody libraries (for discovering highlyspecific binders against a wide variety of antigens) and FGFR3extracellular domain are described below.

Example 1 Attempt to Generate Anti-FGFR3 Antibodies

One hundred micrograms of soluble FGFR3 in complete Freund's Adjuvantwere injected into Balb/c 3T3 naive mice (9 animals). Two repeatedinjections of 20 micrograms were performed at week intervals. 10 daysafter the second booster injection, blood was drawn from animals andserum was tested for the presence of polyclonal antibodies both bymonitoring for binding to the receptor as well as for neutralizingactivity at a dilution of 1:50. No significant neutralizing activity wasobserved in the tested serum (20% at most in some animals). A perfusioninjection of 20 micrograms of soluble receptor was administered 1-2 dayslater but all the mice harboring some activity of neutralizing Ab died.The experiment was repeated twice with the same results.

Example 2 Generation of the FGFR3 Antigens

Two dimeric forms of the extracellular domain of the human FGFR3 wereprepared for use as antigen. One was a histidine-tagged domain with aSerine 371 to Cysteine (S371C) substitution (thanatophoric dysplasia(TD) mutation) to facilitate dimerization and the second one an Fcfusion. The S371C variant was shown to bind heparin and FGF9 coatedplates and to inhibit FGF9-dependent FDCP-FR3 proliferation. The Fcfusion was similarly effective in binding assays, demonstrating itspotential as an inhibitor of FGFR function and as a target for selectingFGFR3 inhibitory molecules. Both soluble receptors were employed toselect neutralizing human recombinant antibodies.

The two variants of the FGFR3 extracellular domain were prepared asfollows:

1. A construct containing the extracellular portion of FGFR3 with athanatophoric dysplasia (TD) mutation to facilitate dimer formationconjugated to a His-tag (histidine tag) was generated. A bluescriptplasmid comprising the human FGFR3 gene (pBS-hFGFR3) was used astemplate for PCR with the following primers:

5′-ACGTGCTAGC TGAGTCCTTG GGGACGGAGC (SEQ ID NO: 2) AG. 5′-ACGTCTCGAGTTAATGGTGA TGGTGATGGT (SEQ ID NO: 3) GTGCATACAC ACA GCCCGCC TCGTC,

wherein the Ser 371 Cys (S371C) substitution is bold and underlined.

The nucleotide sequence encoding the extracellular domain of FGFR3 withthe TD substitution is denoted herein SEQ ID NO:7.

The PCR fragment was digested with XhoI and ligated into pBlueScriptdigested with EcoRV and XhoI. The resulting plasmid, pBsFR3²³⁻³⁷⁴TDhis,was cleaved with NheI and XhoI and the DNA fragment encoding theextracellular domain of FGFR3 was ligated into the same restrictionsites in pCEP-Pu/Ac7 (Yamaguchi et al., 1999; Kohfeldt et al., 1997),generating the pCEP-hFR3²³⁻³⁷⁴TDhis plasmid construct.

To express this FGFR3 variant, 293E cells (EBNA virus transfected 293cells) were transfected with the aforementioned plasmid,pCLP-hFR3²³⁻³⁷⁴TDhis, clones were identified and grown. Cellsupernatants analyzed by Western blot with anti-His antibodydemonstrated high expression of the soluble receptor. Supernatants fromlarge scale preparations were then subjected to batch affinitypurification with Ni-NTA beads and the tagged soluble receptor waseluted by a step gradient ranging from 20 mM to 500 mM imidazol. Asample from each elute was loaded onto a 7.5% SDS-PAGE and stained withGELCODE® (Pierce). In parallel, Western blot analysis was performed andanalyzed with anti-His antibodies. SDS-PAGE (FIG. 1) and immunoblot (notshown) analyses demonstrated peak amounts of purified extracellularFGFR3 in the 2nd (2) 50 mM imidazol fraction. About 0.5 mg of pureprotein was obtained following this single step purification. In FIG. 1,T=total protein, D=dialysed protein, U=unbound fraction.

To assess whether hFR3²³⁻³⁷⁴TDhis (hFR3-TDhis) retained the ability toassociate with heparin and heparin-FGF complex, heparin coated wellswere incubated with 2, 4 or 10 μg purified (labeled as FR3 2, FR3 4 orFR3 10, respectively in FIG. 2) or unpurified (FR3 s) hFR3²³⁻³⁷⁴TDhiswith (checkered bar) or without FGF9 (200 ng/well, hatched bar). Thebinding of hFR3²³⁻³⁷⁴TDhis to each well was determined with anti-Hisantibody. Mock supernatant (M sup), PBS and unpurified mouse FR3AP(FGFR3-alkaline phosphatase, labeled as mFRAP sup) were included ascontrols. The results, as presented in FIG. 2, demonstrated that,similar to what was reported for the wild-type receptor, hFR3²³⁻³⁷⁴TDhis binds to heparin and that this interaction is augmented by thepresence of FGF9. Finally, it was demonstrated that hFR3²³⁻³⁷⁴TDhisinhibits FDCP-FR3 FGF-dependent proliferation in a dose dependentmanner. hFR3²³⁻³⁷⁴TDhis had no inhibitory effect on proliferation whenFDCP-FR3 cells were grown in the presence of IL-3. Taken together,hFR3²³⁻³⁷⁴TDhis proved to be a good candidate as a target antigen forscreening for FGFR3 neutralizing antibodies.

2. The extracellular domain of FGFR3 and FGFR1 were prepared as Fcfusions (FR3exFc and FR1exFc). The amino acid sequence of FGFR3 (NCBIaccess no: NP_(—)000133) is denoted herein SEQ ID NO:1.

To construct the FR3exFc fusion, a polynucleotide sequence (denotedherein SEQ ID NO:4) encoding the extracellular domain of FGFR3 was PCRamplified to contain terminal KpnI and BamHI restriction sites forinsertion into the KpnI and BamHI sites of pCXFc (denoted herein SEQ IDNO:5). This insertion positions the extracellular domain of FGFR3 to beexpressed as a fusion with the Fc amino acid sequence (denoted hereinSEQ ID NO:6).

Both FR3exFc and FR1exFc soluble receptors were demonstrated to beexpressed to a high level in transiently transfected 293T cells (T-cellantigen infected human embryonic kidney 293 cells). The observation thatboth soluble receptors remain bound to heparin-coated wells evenfollowing extensive washes led the laboratory of the present inventorsto try to purify the proteins with the commercial HEPARIN-SEPHAROSE®resin (Pharmacia). One hundred ml volume supernatants, harvested 48hours post-transfection with either FR3exFc or FR1exFc coding plasmids,were incubated overnight at 4° C. with 1 ml HEPARIN-SEPHAROSE® resin.The resin was washed and then subjected to PBS supplemented withincreasing concentration of NaCl. Aliquots of each fraction wereanalyzed by 7.5% SDS-PAGE stained with GELCODE® (Pierce) demonstrating apurification profile of more than 90% homogeneity and a peak elution at400 mM NaCl for FR3exFc (FIG. 3; T=total protein, U=unbound fraction,W=wash). In contrast, FR1exFc was hardly retained on the resin. Thisresult was confirmed by Western analysis of the same fractions withanti-FGFR1ex antibodies demonstrating that most of FR1 exFc is in theunbound fraction (not shown).

Functional analysis of FR3exFc and FR1exFc showed that both competeefficiently for FGF9 binding and stimulating FGFR3, thus, demonstratingtheir potential as inhibitors of FGFRs function and as a target(FR3exFc) for selecting FGFR3 inhibitory molecules.

Neutralizing Effect of Soluble Receptors

The ability of hFR3-TDhis and FR3exFc to inhibit FGF-dependent FDCP-R3cell proliferation was compared. Both soluble receptors inhibitedFDCP-R3 cell proliferation, however, FR3exFc was about 60 times morepotent than hFR3TDhis. Neither had an effect on FDCP cells stimulatedwith IL-3. (FIG. 4; legend: ?-FR3²³⁻³⁷⁴TDhis on FDCP-FR3 cells+FGF9,¦-FR3exFc on FDCP-FR3 cells+FGF9, ?-FR3²³⁻³⁷⁴TDhis on FDCP-FR3cells+IL-3, X-FR3exFc on FDCP-FR3 cells+IL-3). The fact that FR3exFc isentirely in dimeric form whereas only a small proportion ( 1/10) ofhFR3²³⁻³⁷⁴TDhis is in dimeric form might explain, at least in part, thisdifference.

Example 3 Screening for Antibodies

Panning and First Screening of Ab Binding Characterization

The screening strategies to identify Fabs from the Human CombinatorialAntibody Library (HUCAL®, developed at MorphoSys, Munich, Germany anddisclosed in WO 97/08320, U.S. Pat. No. 6,300,064, and Knappik et al.,(2000), the entire contents of which are incorporated herein byreference, using soluble dimeric forms of the extracellular domain ofthe FGFR3 receptor are shown in Table 2.

TABLE 2 Panning Strategies Panning Panning Panning Round 1 Round 2 Round3 Screen 1 FR3-TDhis HEK293 FR3-TDhis Screen 2 FR3exFc RCJ-FR3achFR3exFc captured with captured with mouse anti- mouse anti- human IgGhuman IgG Screen 3 FR3-TDhis RCJ-FR3ach & FR3exFc (Round 1 of RCJ-FR3wtCaptured with panning 1) mouse anti- human IgG

The screening was carried out, for example in Screen 1, by coating thewells of a 96 well plate with hFR3²³⁻³⁷⁴TDhis (FR3-TDhis), panning withthe bacteriophage library and selecting the positive clones. Thepositive clones were then tested on HEK293 (293, human embryonic kidney)cells, expressing endogenous FGFR3. The positive clones were selectedand rescreened on FR3-TDhis. Two additional similar screenings werecarried out as shown in Table 2. In screen 2 the first and thirdpannings were carried out with the FR3exFc antigen and the secondpanning carried out with RCJ cells expressing a mutant (achondroplasia)form of FGFR3. An overview of the number of initial hits and of thecandidate clones is shown in Table 3.

TABLE 3 Overview of Screenings 1, 2 and 3 on FGFR3 consolidated screenedprimary sequenced candidate clones clones hits clones (ELISA & FACS)Screen 1 1076 208 69 15 (MSPRO 1-15) Screen 2 864 300 32 22 (MSPRO 20-33and 52-59) Screen 3 768 487 52 11 (MSPRO 40-50)

Sequence and Vector Data

A plasmid map of the dHLX-MH vector having SEQ ID NO:52 is presented inFIG. 28. FIG. 29 shows the plasmid map of the phage display vector,having SEQ ID NO:53, used in accordance with the present invention.

FIG. 30 displays the polynucleotide sequences of the specific V_(L) andV_(H) domains of MSPRO2 (SEQ ID NO:67 and 77); MSPRO11 (SEQ ID NO:63 and78); MSPRO12 (SEQ ID NO:68 and 82); MSPRO21 (SEQ ID NO:60 and 71);MSPRO24 (SEQ ID NO:57 AND 72); MSPRO26 (SEQ ID NO:64 AND 79); MSPRO28(SEQ ID NO:55 AND 73); MSPRO29 (SEQ ID NO:58 AND 80); MSPRO54 (SEQ IDNO:66 AND 75); MSPRO55 (SEQ ID NO:62 AND 76); and MSPRO59 (SEQ ID NO:69AND 84). The sequences include the framework domains 1-4 and the CDRdomains 1-3. SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:61, andSEQ ID NO:65 denote herein the polynucleotide sequences of the parentV_(L) (kappa or lambda) strands. SEQ ID NO:70, SEQ ID NO:74, SEQ IDNO:81 and SEQ ID NO:83 denote herein the polynucleotide sequences of theV_(H) parent strands.

Example 4 Analysis of Fabs Identified in First Screening

Specificity of Antibody Recognition

The first screening yielded 15 different Fabs that specificallyrecognize FGFR3 in vitro and on the cell surface. Fourteen of these werefurther analysed. LY6.3, an anti-lysosyme antibody, was isolated fromthe same library and serves as a control. ELISA analysis, according tothe following protocol was carried out to determine the specificity ofthe isolated Fabs for FGFR3 or FGFR1.

Fab-FR3/Fc Binding Assay

MAXISORP® ELISA plates were coated with 100 μl anti-human Fc (10 μg/ml)in bicarbonate overnight at 40° C. Wells were washed five consecutivetimes with a PBS solution containing 0.1% Tween 20 (PBST). The wellsurface was blocked with 250 μl PBST+3% BSA (blocking solution) for 1hour at 37° C. This was followed by capturing 1 μg of FGFR/Fc for 1 hourat room temperature. To assess the antibody binding to the capturedFGFR/Fc, 1 μg each of the tested Fabs was incubated in 100 μl blockingsolution per well 1 hour at room temperature. Wells were washed 5 timeswith PBST. Reaction was initiated with the addition of 100 μl of 0.8μg/ml goat anti-human Fab-HRP (horseradish peroxidase) diluted inblocking solution, subsequently washed and detected with TMB substrate(Pierce). The absorbance was measured at 450 nm. A comparison of ELISAanalyses done in both laboratories, Prochon and MorphoSys, is presentedin FIG. 27 and in Table 4.

TABLE 4 ProChon MorphoSys FR1/Fc FR3/Fc FR1/Fc FR3/Fc MS-PRO1 ++ +++/− + MS-PRO2 − ++ − ++ MS-PRO3 + ++ − ++ MS-PRO4 − + − ++ MS-PRO5 − +++/− + MS-PRO6 − ++ − + MS-PRO7 − ++ − + MS-PRO8 + ++ − + MS-PRO9 − +/−+/− + MS-PRO10 + ++ − ++ MS-PRO11 − +/− + ++ MS-PRO12 − +/− − ++MS-PRO13 − +/− +/− + MS-PRO14 − − − + LY6.3 (control) − −

In most cases, the data generated in both laboratories are in agreement.However, some Fabs behave differently. For example, MS-PRO3 andMS-PRO-10 were found to be completely FGFR3-specific under certainconditions while under other conditions both show considerablecross-reaction with FGFR1. Subsequent FACS analysis supported the crossreactivity for MS-PRO3, but not for MS-PRO10. Taking into account thepotency and specificity of the Fabs, MS-PRO2 had the highest scoreaccording to these preliminary data.

Example 5 Affinity of Fab to FGFR3

The affinity measurements were performed by BIACORE® analysis accordingto the standard procedure recommended by the supplier (Pharmacia). Theanti-Fc antibody was coupled via the EDC/NHS chemistry to the chip andsubsequently FGFR3 was captured. The Fabs of the invention were thenbound to this surface.

Table 5 shows a comparison of affinities of Fabs candidates to FGFR3 asdetermined by BIACORE® and by FACS-scatchard.

TABLE 5 Comparison of Antibody Affinities to FGFR3 determined byBIAcore ® and FACS-Scatchard BIAcore ® Indirect FACS-Scatchard Fab clone[nM] [nM] MSPRO2 37 ± 10 43 MSPRO11 4 ± 2 4 MSPRO12 14 ± 2  6.5 MSPRO219 ± 2 0.6 MSPRO24 10 ± 2  0.3 MSPRO26 4 ± 1 1.4 MSPRO28   9 ± 0.4 0.3MSPRO29 6 ± 4 0.4

Table 1E (in the Detailed Description, vide supra) shows the affinity asdetermined by BIACORE® for the Fab candidates shown in Table 5 convertedinto the Fab mini-antibody format, Fab-dHLX-MH, where a dimer of the Fabmonomer is produced after insertion into an expression vector as afusion protein.

Table 6 shows the results of a competition assay wherein each MSPRO Fabwas bound to FGFR3 at a concentration of 500 nM or 1,000 nM andcoinjected in pairs with the other MSPRO Fabs. The (−) indicates bindingto the same or nearby epitope while (+) indicates binding to differentepitope. The results show that MSPRO2 and 12 bind to the same or nearbyepitope while MSPRO 11, 21, 24, 26, 28 and 29 bind to an epitopedifferent from that of MSPRO 2 or 12.

TABLE 6 2 11 12 21 24 26 28 29 2 + − + + + + + 11 + + − − − − − 12− + + + + + + 21 + − + − − − − 24 + − + − − − − 26 + − + − − − − 28 +− + − − − − 29 + − + − − − −

Example 6 Specific Neutralizing Activity of the Antibodies

A: FDCP Cell Proliferation Assay

The FDCP cell line is a murine immortalized, interleukin 3 (IL-3)dependent cell line of myelocytic bone marrow origin, which does notexpress endogenous FGF Receptors (FGFR). Upon transfection with FGFRcDNA, the FDCP cell line exhibits an FGF dose-dependent proliferativeresponse that can replace the dependence on IL-3. FDCP cell lines,transfected with FGFR cDNAs can therefore be used to screen for specificinhibitors or activators of FGFR, as well as for analyzing FGFRsignaling. The FDCP cell response to various ligands was quantitated bya cell proliferation assay with XTT reagent (Cell Proliferation Kit,Biological Industries Co.). The method is based on the capability ofmitochondrial enzymes to reduce tetrazolium salts into soluble coloredformazan compounds which can be quanititated and is indicative of cellviability. Specifically, FDCP cells expressing FGFR3IIIb, FGFR3IIIc orFGFR1 were grown in “full medium” (Iscove's Medium containing 2 mlglutamine, 10% FCS, 100 ug/ml penicillin, 100 ug/ml streptomycin)supplemented with 5 ug/ml heparin and 10 ng/ml FGF9. Cells were splitevery 3 days and kept in culture no more than one month. One day priorto the experiment, the cells were split. Before the experiment, thecells were washed 3 times (1000 rpm, 6 min) with full medium. Later, thecells were resuspended and counted with Trypan Blue. Twenty thousand(20,000) cells per well were added to wells in a 96-well plate in 50 ulin full medium containing 5 ug/ml heparin. Conditioned medium was addedin an additional volume of 50 ul full medium containing FGF9 at varyingconcentrations to a final volume of 100 ul. A primary stock solution(usually 2× the higher concentration) of the antibody (or Fabs) wasprepared in Iscove's+++ containing 5 μg/ml heparin and 2.5 ng/ml FGF9 orIL-3 (final concentration 1.25 ng/ml). Dilutions were filtered in a 0.2μm syringe nitrocellulose filter blocked first with 1 mg/ml BSA andwashed then with Iscove's+++. Aliquots of requested serial dilutionswere prepared. Dilutions were kept on ice until use. 50 μl of thecorresponding 2× final concentration was added to each well and theplate was incubated at 37° C. for either 40 hours or 64 hours. Afterincubation, the reaction was developed as follows: 100 μl of activatorsolution was added to 5 ml XTT reagent and mixed gently. 50 μl ofmixture was added to each well. Optical density (OD) at 490 nm at thispoint gave the zero time reading.

Cells were then incubated at 37° C. for 4 hours (in the case of a40-hour incubation) or 2 hours (in the case of a 64-hour incubation) andproliferation was measured by O.D. at 490 nm (A490).

It is noted that the assay is successful when the O.D. of untreatedcontrol growing with saturated amounts of FGF (10 and 20 ng/ml) is atleast 1.3 O.D. units. Furthermore, it is noted that the background ofwells with no cells should be 0.2-0.35 O.D. units and that the O.D.absorbance of 1.25 ng/ml FGF9 should not be less than 40% of the O.D.absorbance achieved with saturated FGF 9 concentration (10 and 20ng/ml). Specific inhibition of FGF and FGF receptor mediatedproliferation should always be accompanied with lack of any inhibitionof the same antibody concentration on IL-3 dependent cell proliferation.

The following FDCP cell lines were used:

*FDCP-C10 (C10): FDCP cells transfected with the human wild-typeFGFR3IIIc.

*FDCP-R3: FDCP cells transfected with the human wild-type FGFR3IIIb.

*FDCP-R1: FDCP cells transfected with the human wild-type FGFR1.

*FDCP-F3Ach: FDCP cells infected with human FGFR3 mutated at amino acidGlycine 380 to Arginine (G380R), analogous to the most common humanachondroplasia mutation.

B: Neutralizing Activity

The neutralizing activity of the antibodies was measured by theaforementioned cell proliferation analysis in FDCP-FR3 and FDCP-FR1 celllines and is presented in FIG. 5. Increasing amounts of the indicatedFabs (MSPRO 2, 3 and 4) were added to FDCP-FR3 (closed triangle ? (2),star * (3), and circle ? (4)) or FDCP-FR1 (open triangle ? (2), opensquare ? (3) and open circle ? (4)) grown in the presence of FGF9. Twodays later, an XTT proliferation assay was performed. While none of theFabs inhibited FDCP-FR1 cell proliferation, MSPRO2 (?) and MSPRO3 (*)inhibited FDCP-FR3 proliferation with a similar IC50 of about 1.0 μg/ml.In contrast, MSPRO4 (?) had no inhibitory effect on FDCP-FR3proliferation. The rest of the Fabs, MSPRO 1, 3, 5, 6, 7, 9, 11, 12, 13,14, were similarly analyzed on FDCP-FR3 expressing cells. Increasingamounts of the indicated Fabs were added to FDCP-FR3 grown in thepresence of FGF9 (FIG. 6). Inhibitors of FGFR3 signaling were antibodiesMSPRO 1, 3, 5, 7, 9, 11, 12. The results of the proliferation assay doneat two sites are compared in Table 7 (NA=data not available).

TABLE 7 Prochon MorphoSys FDCP-FR1 FDCP-FR3 FDCP-FR1 FDCP-FR3 MSPRO1 −++ NA NA MSPRO2 − ++ NA ++ MSPRO3 − ++ NA ++ MSPRO4 − − NA − MSPRO5 − +NA + MSPRO6 − − NA +/− MSPRO7 − ++ NA + MSPRO8 − +/− NA +/− MSPRO9 − +NA + MSPRO10 − + NA NA MSPRO11 − +++ NA ++ MSPRO12 − +++ NA +++ MSPRO13− − NA NA MSPRO14 − − NA NA LY6.3 − − NA NA

As shown in Table 7, there is an excellent agreement between the data.About half of the Fabs show considerable neutralizing activity, MSPRO12being the most potent. Most of the inhibitory Fabs performed well in thebinding assay (Table 4), with MSPRO11 and MSPRO12 being the exception tothe rule, however, clearly remain good candidates to pursue. None of theFabs (including those that crossreact with FGFR1) inhibitedFGF-dependent FDCP-FR1 proliferation. In addition, FDCP-FR3 cells grownin the presence of IL-3 were not affected by any of the Fabs.

An additional 20 new Fabs were selected from the second panning. Threeof these new Fabs were subjected to the FDCP cell proliferation test andall were found to neutralize the receptor (MSPRO52 (?), MSPRO54 (?) andMSPRO55 (*) in FIG. 7A). Interestingly and in accord with MorphoSysaffinity data, one Fab (MSPRO54) showed strong neutralizing activityagainst FGFR1 (FIG. 7B). MSPRO29 (?) and a control antibody Ly6.3 (¦)were also tested in this assay.

Example 7 Receptor Expression and Activation in RCJ Cells

RCJ Cell Assay

RCJ cells (fetal rat calvaria-derived mesenchymal cells, RCJ 3.1C5.18;Grigoriadis, 1988) were generated to express various FGF Receptors in aninducible manner, in the absence of tetracycline. The M14 line(RCJ-FR3ach) expresses FGFR3-ach380 mutant upon induction by the removalof tetracycline. The cells were incubated in low serum after which FGFwas added to stimulate receptor function and signaling. The cells werelysed and the receptor level, receptor activation and signaling areassessed by Western with anti-active ERK (or JNK) (Promega). The lysatesis immunoprecipitated with anti-FGFR3 (Santa Cruz), and a Westernimmunoblots is prerformed using anti-phospho-tyrosine (Promega)antibodies. W11 refers to the RCJ cells expressing wild type FGFR3.RCJ-FR1 and RCJ-FR2 refer to RCJ cells expressing the FGFR1 and FGFR2receptors, respectively. FIG. 21 provides a flow chart of theexperimental procedure.

The transfected RCJ cells were grown in a-MEM supplemented with 15%fetal calf serum, 1× penicillin/streptomycin/nystatin, 1× glutamine, 600μg/ml neomycin, 2 μg/ml tetracycline, 50 μg/ml hygromycin B tosubconfluence. The medium was aspirated off and the cells washed withtrypsin, 1 ml/10 cm dish, then trypsinized with 0.5 ml/10 cm dish. Thecells were resuspended in 10 ml a-MEM supplemented with 15% fetal calfserum, 1× penicillin/streptomycin/nystatin, 1× glutamine, 600 μg/mlneomycin, and 2 μg/ml tetracycline.

Six hundred thousand (6×10⁶) cells/well were seeded in a 6-well dish.The cells were washed thrice 24 hours later (or 8 hours later if twicethe amount of cells are seeded) with 1 ml a-MEM, and then incubated witha-MEM supplemented with 15% fetal calf serum, 1×penicillin/streptomycin/nystatin, and 1× glutamine (induction medium)for 16 hours. Cells were washed thrice with 1 ml a-MEM and allowed togrow for 4 additional hours in 1 ml of 0.5% exhausted serum (prepared bydiluting the induction medium X30 with a-MEM).

FGF9 (1 ng/ml) was added for 5 minutes and the cells placed on ice. Thecells were washed twice with ice-cold PBS and lysed with 0.5 ml lysisbuffer. The cells were scraped into an Eppendorf tube, vortexed once andplaced on ice for 10 minutes. The lysate was microcentrifuged for 10minutes at 4° C., and the cleared lysate was transferred into a freshEppendorf tube.

The protein content was determined by Bradford or DC protein assay(Bio-Rad, cat# 500-0116) following manufacture instructions. Totalprotein aliquots, supplemented with 1/5 volume of 5× sample buffer, wereboiled for 5 minutes and stored at −20° C. until ready to load on gel.In parallel an immunoprecipitation (IP) assay was performed, 10 μlanti-FGFR3 antibodies were added to the rest of the lysates andincubated for 4 hours at 40° C. Twenty (20) ul protein A-SEPHAROSE® wasadded and incubated for 1 hour at 4° C. with continuous shaking.Afterwards, the mixture was microcentrifuged 15 seconds, and the fluidwas aspirated, carefully leaving a volume of ˜30 μl above the beads. Thebeads were washed 3 times with 1 ml lysis buffer. At this step, theprotease inhibitor mix was omitted from the buffer.

After the final wash, 15 μl of 5× sample buffer was added, samples wereboiled 5 minutes and stored at −20° C. until ready to load onto gel.Samples were loaded onto a 7.5% SDS-PAGE, cast on a Mini-PROTEAN IIelectrophoresis cell, and run at 100 V through the upper gel and at 150V through the lower gel. Proteins were transferred onto a nitrocellulosesheet using the Mini trans-blot electrophoretic transfer cell at 100 Vfor 75 minutes or at 15 V overnight. The lower part of the total lysateWestern blots was probed with anti-active JNK (anti-phosphorylated JunKinase) and the upper part was probed with anti-FGFR3, both at 5×10³dilutions.

FIG. 8A shows that MSPRO2 blocks FGFR3 activation in W11 cells andweakly blocks signaling in M14 cells, and MSPRO12 blocks FGFR3 receptoractivation in W11 and M14 expressing cells. Furthermore MSPRO13 appearedto be able to block FGFR1 activation while none of the Fabs blockedFGFR2 activation. FIG. 8B shows the inhibitory capacity of MSPRO12 andMSPRO59 on wild type FGFR3 expressing cells, as seen as reduction in JNKsignaling. MSPRO29 strongly inhibits FGFR3 activation (<5 ug), MSPRO12has an inhibitory effect but at a higher concentration (5-20 ug).

The IP lysate Western blots were probed with anti-phosphotyrosine (R&DSystems). Hybridization was detected by ECL following the manufacturer'sinstructions.

BIACORE® and proliferation analyses showed that among the new Fabs,MSPRO54 is highly cross reactive with FGFR1. To further test the crossreactivity of the new Fabs, RCJ cells expressing either FGFR3ach(RCJ-M14; M14 on FIG. 9A) FGFR3 wild type (W 11 on FIG. 9B), FGFR1 (R1-1on FIG. 9C) or FGFR2 (R2-2 on FIG. 9D) were incubated with increasingamount of a control antibody LY6.3, MSPRO29, 54 and 59 for one hour.FGF9 was added for 5 minutes and cell lysates were analyzed by Westernblot for ERK activation (phosphorylated ERK; pERK) (FIGS. 9A, 9B, 9C and9D). Furthermore, MSPRO13 was able to block FGFR1 activation while noneof the Fabs blocked FGFR2 activation. FIGS. 9A. 9B, 9C and 9D show theresults of several Fabs, at different mg concentrations, on RCJexpressing wildtype FGFR3 or the different FGFR types. MSPRO29 appearedas the best FGFR3 blocker and was also effective in blocking FGFR1 (FIG.9 c); however, MSPRO54 was the most effective Fab against FGFR1. None ofthe Fabs significantly inhibited FGFR2 activity. There are only a fewamino acid residues within the third Ig domain that are shared by FGFR3and FGFR1 but not by FGFR2. Making mutants at these sites should clarifytheir role in Fab-receptor binding.

Example 8 Epitope Mapping of Selected Fabs

Constructs comprising cDNAs that code for segments of the extracellulardomain of FGFR3 were generated (FIG. 10). D2 comprises Ig domain 2, D2-3comprises Ig domains 2 and 3, and D1-3 comprises Ig domains 1, 2 and 3.These constructs include pChFR3^(D2)Fc that codes for Ig-like domain 2of FGFR3 and pChFR3^(D2,3)Fc that codes for domain 2 and 3, both ashuman Fc fusions. The corresponding chimeric proteins, as well as thecontrol hFR3exFc (containing domains 1, 2 and 3) were anchored to anELISA plate coated with a human Fc antibody. A panel of 8 best Fabs,MSPRO2, 11, 12, 21, 24, 26, 28 and 29, were added, and bound Fab wasdetermined with HRP-a human Fab (FIG. 11). The results in FIG. 11demonstrate that MSPRO2 (speckled bar) and MSPRO12 (diagonally hatchedbar) differ from the other tested Fabs. Both bind to the Ig like domain2 while the others require domain 3 for binding. It was then testedwhether or not Fabs that belong to the second group would distinguishthe FGFR3IIIc isoform from the FGFRIIIb from. FDCP-FR3IIIb orFDCP-FR3IIIc cells were incubated in the presence of 1.25 ng/ml FGF9with increasing doses of either MSPRO12 or MSPRO29. Ly6.3 was includedas control. After 2 days in culture, cell proliferation was measuredwith the XTT reagent. Clearly, MSPRO29 (open triangle) was completelyineffective against the IIIb isoform (FIG. 12). In contrast, MSPRO12(square on hatched or solid lines) was equally effective against bothisoforms. These data suggest that residues that differ between the twoisoform are critical for MSPRO29 (and probably also for the other Fabsin the same group) FGFR3 binding.

Domains in FGFR3 Recognized by the New Fabs

From the data presented, MSPRO antibodies can be divided into 2 groups,one that includes Fabs that bind the FGFR3 Ig II domain (MSPRO2 and 12)and a second with members that require the Ig III domain for binding(MSPRO11, 21, 24, 26, 28, and 29). To classify the new Fabs obtainedfrom the last screen, as well as some previously obtained Fabs, aproliferation assay of FDCP cells expressing either FR3IIIb or FR3IIIcwas performed. The cells were incubated in the presence of 10 (IIIb) or5 (IIIc) ng/ml FGF9 with increasing doses of the indicated Fabs. After 2days in culture, cell proliferation was measured with the XTT reagent.

The data shows that MSPRO59 (*) efficiently inhibited both FDCP-FR3IIIb(FIG. 13A) and FDCP-FR3IIIc cells (FIG. 13B), while MSPRO21, 24, 26, 28,29 and 54 inhibited FDCP-FR3IIIc proliferation only.

Example 9 Bone Culture

Radiolabeled MSPRO29 was used to determine whether MSPRO Fabs are ableto penetrate the bone growth plate.

To determine the effect of iodination on Fab activity, 50 μg of MSPRO29was first labeled with cold iodine using Pierce IodoGen coated tubes.The process was carried out either without iodine, with 0.04 mM NaI (lowI) or with 1 mM NaI (high I). MSPRO29 was then purified through aSEPHADEX® G-50 column. The ability of the modified Fab to bind FGFR3 wasdetermined by ELISA. MAXISORP® wells were coated with anti-human Fc.FGFR3/Fc was then anchored to the wells. In parallel, a similar set ofwells was left in blocking buffer only (no FR3/Fc, hatched bars). Theunmodified (no I) or the modified MSPRO29 (low or high, 2 G-50 fractionseach, 1 and 2) were added at approximately 5 μg/well and binding wasmeasured with anti-human Fab. Fresh MSPRO29 and buffer alone wereincluded as controls (FIG. 14: FGFR3/Fc, checkered bars; no FGFR3/Fc,hatched bars). MS-PRO29 was labeled with 1 mCi ¹²⁵I. The specificactivity of the Fab was 17 μCi/μg.

MSPRO29 labeled in the presence of 0.04 mM NaI showed equal binding tothe receptor as compared to the control unmodified Fab. MSPRO29 labeledin the presence of 1 mM NaI (high I, 1 and 2) also bound the receptor,however, the noise level of this sample was as high as the signal itselfsuggesting that at the high iodide concentration the Fab wasinactivated.

The neutralizing activity of the modified Fab was tested in aproliferation assay using FDCP-FR3 (C10) (FIG. 15). FDCP-FR3 (C10) cellswere treated with the indicated amount of labeled or unlabeled (withoutI) MSPRO29. The proliferation rate of the cells was determined by XTTanalysis. The Fab was labeled at either 0.04 mM (Low) or 1 mM NaI(High). Two G-50 fractions (1 and 2) were analyzed. Fresh MSPRO29 andbuffer alone (mock) were included as controls.

This experiment showed that MSPRO29, labeled at 0.04 mM NaI, maintainedits inhibiting activity almost entirely while MSPRO29 labeled at 1 mMNaI had indeed lost its activity completely.

Ex Vivo Distribution of ¹²⁵I MSPRO29 in Bone Culture

Femora prepared from newborn mice were incubated with 2 μg ¹²⁵I-MSPRO29(17 μCi/μg) or ¹²⁵I-Ly6.3 (20 μCi/μg) for 1, 3 or 5 days in culture.Then, sections were processed for radiomicroscopy. After 3 days inculture, MSPRO29 was predominantly visualized at the higher hypertrophiczone and to a lesser extent at the secondary ossification region FIGS.16A, 16B 16C, 16D, 16E and 16F). Hematoxylin-eosin staining of growthplate treated with radiolabeled MSPRO29 or Ly6.3 (FIGS. 16A and 16D,respectively) ×100 magnification. Radiomicroscopic sections of growthplate treated with radiolabeled MSPRO29 or Ly6.3 (FIGS. 16B and 16E) at×100 magnification. FIGS. 16C and 16F are the same as FIGS. 16B and 16Ebut at ×400 magnification. The arrows in FIGS. 16B and 16C indicate thelocation of the specific binding of the radiolabelled MSPRO29 to thehigher hypertrophic zone of the growth plate.

As compared to MSPRO29, the control Ly6.3 Fab was weakly and evenlydistributed throughout the whole growth plate. At day 1 in culture, thesignal was weaker but with similar distribution pattern. Thisdistribution also holds at 5 days in culture with a less favorablesignal to noise ratio (data not shown). This clearly demonstrates thatMSPRO29 binds FGFR3 in our target organ.

Example 10 Neutralization of Constitutively Active Receptors

The inhibitory activity of MSPRO antibodies on ligand-dependent andligand-independent FDCP proliferation expressing FGFR3 Achondroplasiamutation was tested.

A proliferation assay was carried out using FDCP-FR3wt (C10, FIG. 17A)or FDCP-FR3ach cells (FIG. 17B) incubated with 1.25 or 5 ng/ml FGF9respectively and with increasing amounts of MSPRO54 or MSPRO59. As shownin FIG. 17, both MSPRO54 (diamond ?) and 59 (square ¦) antibodiesneutralize the mutant receptor. FDCP-FR3ach acquired ligand independentcell proliferation due to the high expression of the FGFR3ach mutation.MSPRO29 (?) inhibits the FDCP-FR3wt activity at a level similar toMSPRO54 and 59 but is less effective in inhibiting the FGFR3ach receptorin this assay system.

FDCP cells that express the achondroplasia FGFR3 (FDCP-FR3ach) andproliferate independently of ligand were incubated with the indicatedamount of MSPRO12, 29, 59 or the control Ly6.3. Two days later, cellproliferation was determined by an XTT analysis. When inhibition of cellproliferation by the MS-PRO 12, 29, 54 and 59 were tested, only theantibodies 12 and 59 (the only Ab which recognized D2 domain) inhibitedthe ligand-independent cell proliferation (FIGS. 18A and 18B).

Example 11 RCS Chondrocyte Culture

Effect of Fabs on Growth Arrest of RCS Chondrocytes

RCS is a rat chondrosarcoma derived cell line expressing preferentiallyhigh levels of FGFR2 and FGFR3 and low levels of FGFR1 (Sahni, 1999). Inthis cell line FGFR functions as an inhibitor of cell proliferationsimilar to its expected role in the achondroplasia phenotype. Analysisof RCS cell proliferation mediated by the addition of differentmolecules of the invention, showed that MSPRO54 and MSPRO59 were able torestore cell proliferation.

The screening was performed on RCS parental cells in 96 well plates.Cells were seeded at a concentration of 2,000 cells/well. The followingday 10 ng/ml FGF-9 and 5 μg/ml heparin were added to the cells. 50 ug/mlof the antibodies were added. Positive and negative controls for cellproliferation are included in this assay at the same concentrations asthe tested molecules. On the fourth day of incubation, plates wereobserved under the microscope. If all cells were viable, no quantitativeassay to measure the effect of the variants was performed. If cell deathwas observed, the Cy-Quant assay kit is used to measure the amount ofthe cells. The results are measured in a fluoro ELISA reader. FIG. 19shows the ELISA results in bar graph form. Untreated cells are shownspeckled, ligand treated cells are shown in gray, control antibody(LY6.3) treated cells are in black while MSPRO54 and MSPRO59 treatedcells are shown in diagonally hatched or checkered bars, respectively.

Example 12 Ex Vivo Bone Culture

The femoral bone cultures were performed by excising the hind limbs of369-mice, heterozygous or homozygous mice for the achondroplasia G369Cmutation (age P0). The limbs were carefully cleaned up from thesurrounding tissue (skin and muscles) and the femora exposed. The femorawere removed and further cleared from tissue remains and ligaments. Thefemora were measured for their initial length, using a binocular with aneyepiece micrometer ruler. The bones were grown in 1 ml of medium in a24 well tissue culture dish. The growing medium is a-MEM supplementedwith penicillin (100 units/ml), streptomycin (0.1 mg/ml) and nystatin(12.5 units/ml). In addition, the medium contains BSA (0.2%),a-glycerophosphate (1 mM) and freshly prepared ascorbic acid (50 μg/ml).The bones were cultured for 15 days. Measurements of bone length andmedium replacement were performed every three days.

At the end of the experiment, the growth rate of the bones wasdetermined. The growth rate of bones is calculated from the slope of alinear regression fit on the length measurements obtained from day 3 to9.

The result, as shown in FIG. 20, demonstrate a dose dependent increasein the growth rate of bones treated with MS-PRO 59 in comparison tonon-relevant control LY6.3 Fab. The LY6.3-treated control femurs, markedwith a circle, grew at the slowest rate. The MSPRO59 treated femursexhibited a higher growth rate, with the optimal rate achieved at thehighest MSPRO59 concentration of 400 ug/ml (square), which can be seenas the steeper slope. Moreover, the growth rates achieved by 400microgram/ml of MSPRO59 doubled in comparison to the control Ab (3.55U/day as compared to 1.88 U/day, respectively). This experiment showsthe neutralizing effect of the MSPRO59 antibody on an ach mutant FGFR3,in an ex vivo model.

Example 13 In-vivo Trials

FDCP-FR3ach cells, but not FDCP (control) cells, were found to betumorigenic when injected into nude mice. Each of 9 mice received twosub-cutaneous injections with different amount of transfected cells.Fourteen days after injection, progressively growing tumors started toappear at the site of FDCP-FR3ach injection but not at the FDCP site ofinjection. External examination of the tumors showed a high vascularcapsule. ¹²⁵I-labeled MSPRO59 and LY6.3 were injected I.P. into nudemice carrying the FDCP-FR3ach derived tumor. The tumors were dissected 4and 24 hours later and radioactivity was measured. Concentration oflabeled MSPRO59 Abs in FDCP-FR3ach derived tumors is shown in FIG. 22.

Example 14 Animal Model for Bladder Carcinoma

Recent studies have shown that the IIIb isoform of FGFR3 is the onlyform expressed in bladder carcinoma, in particular an FGFR3 with anamino acid substitution wherein Serine 249 is replaced by Cysteine(S249C). The progression of the cancer is believed to be a result of theconstitutive activation resulting from this amino acid substitution. Inorder to create the FGFR3 IIIb form, we isolated the IIIb region ofFGFR3 from HeLa cells and generated a full length FFGR3IIIb isoform inpLXSN. Retroviruses, expressing either normal FGFR3 (FR3wt) or mutantFGFR3 (FR3-S249C) were produced and used to infect FDCP cells. Stablepools were generated and further used for in-vitro and in-vivoexperiments.

MSPRO59 Reduces Tumor Size in Mice

Twelve nude mice were injected with 2×10⁶ FDCP-S249C cells subcutaneousat 2 locations, one on each flank. A week later MSPRO59 was administeredI.P. at 400 ug per mouse (3 mice in total), followed by 3 injections of275 ug each, in 2 to 3 days intervals. Following 24 and 26 days thetumor size was measures. FIG. 23 shows the inhibitory effect of MSPRO59on tumor size.

Treatment of FDCP-S249C-derived Tumors with MSPRO59

Nude mice (3 in each group), were injected subcutaneous at 2 locations,one on each flank, with 2×10⁶ FDCP-S249C cells each. A week later, 400or 80 μg MSPRO59 were injected IP. Three days later, mice were injectedwith 400 μg followed by 5 additional injections with 275 μg MSPRO59,each, every 3 or 4 days. Mice initially treated with 80 μg MSPRO59 weresimilarly given an additional 80 μg MSPRO59 followed by 5 injectionswith 50 μg MSPRO59 at the same schedule. Mice injected with PBS wereused as control. Tumor volume was estimated from measurements in 3dimensions at 16,20, 23 or 32 days post cell injection. As shown in FIG.24 there is both a delay in tumor appearance and an inhibitory effect ontumor progression in the treated mice. This indicates that these FGFR3inhibitors are potent in vivo.

These data may also help us understand the mechanism by which theS249C-derived tumors were developed. Since we are using pools of cells,treatment with MSPRO59 inhibited the susceptible cells, leading to delayin tumor appearance. However, over time, the resistant cells survivedand proliferated, giving rise to a solid tumor.

MSPRO59 Inhibits FDCP-FR3ach380 Derived Tumor Growth.

Nude mice were injected subcutaneously in the flank with 2×10⁶FDCP-FR3ach380 cells, each. Treatment with MSPRO59 began at the day oftumor appearance. Three mice were treated with a known tyrosine kinaseinhibitor (TKI −50 mg/Kg/injection) and three with 400 μg followed by 3additional injections with 300 μg MSPRO59, every 3 or 4 days. Three micewere treated with PBS alone as control. The tumor size was estimated asbefore at the indicated days after cell injection. The dose schedule isshown in Table 8 below.

TABLE 8 Days After FDCP-FR3^(ach380) Cell Injection 21 25 28 31 MSPRO59(μg) 400 μg 300 μg 300 μg 300 μg PBS (μl)  50  50  50  50

Results are shown in bar graph format in FIG. 25A.

MSPRO59 Inhibits FDCP-S249C Induced Tumor Growth

After several months in culture FDCP-S249C cells acquire partialresistance to MSPRO antibodies and eventually become completelyinsensitive. To overcome the instability of the FDCP-derived pools,clones from a pool of FDCP-S249C cells were isolated and characterized.These clones were tested in an XTT proliferation assay and were shown tobe inhibited by MSPRO59. 2×10⁶ cells from each clone were injected intonude mice. Tumors appeared 18-30 after injection.

FDCP-S249C clone cells were injected subcutaneously on the flank. A weeklater mice were injected with 280 μg MSPRO59 single chain (SC) I.P.every day. Mice injected with PBS were used as control. Tumor volume wasestimated from measurements in 3 dimensions at 18 or 24 days post cellinjection. An apparent inhibition of tumor growth by MSPRO59 (ScFv) wasobserved in tumors derived from clone 2 (FIG. 26). FIG. 25B shows theinhibition effected by MSPRO59scFv and MSPRO59 Fab compared to thecontrol. Both inhibit growth of the tumor resulting from constitutivelyactivated cells.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by references. Reference to known methodsteps, conventional methods steps, known methods or conventional methodsis not in any way an admission that any aspect, description orembodiment of the present invention is disclosed, taught or suggested inthe relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

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1. An isolated antibody or antigen-binding fragment thereof, whereinsaid antibody or said antigen-binding fragment thereof specificallybinds the extracellular domain of the human fibroblast growth factorreceptor 3 (FGFR3) polypeptide comprising the amino acid sequence of SEQID NO:1 and wherein said antibody or said antigen-binding fragmentthereof comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO:106 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:95.
 2. A compositioncomprising the antibody or antigen-binding fragment thereof according toclaim
 1. 3. A kit comprising the antibody or antigen-binding fragmentthereof of claim
 1. 4. An isolated antibody or antigen-binding fragmentthereof, wherein said antibody or said antigen-binding fragment thereofspecifically binds the extracellular domain of the human fibroblastgrowth factor receptor 3 (FGFR3) polypeptide comprising the amino acidsequence of SEQ ID NO:1 and wherein said antibody or saidantigen-binding fragment thereof comprises a heavy chain variable regioncomprising the 3 complementarity determining region (CDR) amino acidsequences of the heavy chain variable region amino acid sequence of SEQID NO:106 and a light chain variable region comprising the 3 CDR aminoacid sequences of the light chain variable region amino acid sequence ofSEQ ID NO:95.
 5. A composition comprising the antibody orantigen-binding fragment thereof according to claim
 4. 6. A kitcomprising the antibody or antigen-binding fragment thereof of claim 4.